Monocyclic heteroaryl compounds

ABSTRACT

This invention relates to compounds of the general formula: 
     
       
         
         
             
             
         
       
         
         
           
             in which the variable groups are as defined herein, and to their preparation and use.

BACKGROUND OF THE INVENTION

The protein kinases represent a large family of proteins, which play acentral role in the regulation of a wide variety of cellular processesand maintaining control over cellular function. A partial, non limiting,list of such kinases includes abl, Akt, bcr-abl, Blk, Brk, c-kit, c-met,c-src, CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, CDK10,cRaf1, CSK, EGFR, ErbB2, ErbB3, ErbB4, Erk, Pak, fes, FGFR1, FGFR2,FGFR3, FGFR4, FGFR5, Fgr, flt-1, Fps, Frk, Fyn, Hck, IGF-1R, INS-R, Jak,KDR, Lck, Lyn, MEK, p38, PDGFR, PIK, PKC, PYK2, ros, tie, tie2, TRK andZap70. Abnormal protein kinase activity has been related to severaldisorders, ranging from non-life threatening diseases such as psoriasisto extremely serious diseases such as cancers.

In view of this large number of protein kinases and the multitude ofprotein kinase related diseases, there is an ever-existing need toprovide new classes of compounds with increased selectivity that areuseful as protein kinase inhibitors and therefore useful in thetreatment of protein tyrosine kinase related diseases.

This invention concerns a new family of acetylenic heteroaryl compoundsand their use in treating cancers, bone disorders, metabolic disorders,inflammatory disorders and other diseases.

DESCRIPTION OF THE INVENTION 1. General Description of Compounds of theInvention

The compounds of this invention have a broad range of useful biologicaland pharmacological activities, permitting their use in pharmaceuticalcompositions and methods for treating metabolic disorders, bone diseases(e.g., osteoporosis, Paget's Disease, etc.), inflammation (includingrheumatoid arthritis, among other inflammatory disorders) and cancer(including solid tumors and leukemias, especially those mediated by oneor more kinases such as Src or kdr, or by dysregulation of a kinase suchas Abl and mutant variants thereof), including, among others, advancedcases and cases which are resistant or refractory to one or more othertreatments.

Included are compounds of Formula I, a tautomer, an individual isomer, amixture of isomers or a pharmaceutically acceptable salt, solvate orhydrate thereof:

Ring T represents a 5-membered monocyclic heteroaryl ring, comprising1-3 heteroatoms selected from O, N, S and being optionally substitutedwith 1-3 R^(t) groups;

Ring A represents a 5- or 6-membered aryl or heteroaryl ring and isoptionally substituted with 1-4 R^(a) groups;

Ring B represents a 5- or 6-membered aryl or heteroaryl ring and isoptionally substituted with 1-5 R^(b) groups;

L¹ is selected from NR¹C(O) and C(O)NR¹;

At each occurrence, R^(a), R^(b) and R^(t), is independently selectedfrom the group consisting of halo, —CN, —NO₂, —R⁴, —OR², —NR²R³,—C(O)YR², —OC(O)YR², —NR²C(O)YR², —SC(O)YR², —NR²C(═S)YR², —OC(═S)YR²,—C(═S)YR², —YC(═NR³)YR², —YP(═O)(YR⁴)(YR⁴), —Si(R⁴)₃, —NR²SO₂R²,—S(O)_(r)R², —SO₂NR² _(R) ³ and —NR²SO₂NR² _(R) ³, wherein each Y isindependently a bond, —O—, —S— or —NR³—;

R¹, R² and R³ are independently selected from H, alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic andheteroaryl;

alternatively, NR²R³ moiety may be a 5- or 6- membered saturated,partially saturated or unsaturated ring, which can be optionallysubstituted and which contains 0-2 additional heteroatoms selected fromN, O and S(O)_(r);

each occurrence of R⁴ is independently selected from alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic andheteroaryl;

each of the foregoing alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, aryl, heteroaryl and heterocyclic moiety is optionallysubstituted;

m is 0, 1, 2, 3 or 4;

n is 0, 1, 2 or 3;

p is 0, 1, 2, 3, 4 or 5; and

r is 0, 1 or 2.

The foregoing definitions are further elaborated upon and exemplifiedbelow and apply to all subsequent occurrences except to the extentotherwise specified.

2. Featured Classes of Compounds and Their Use, Generally

In compounds of this invention, Ring T is optionally substituted on oneto three ring atoms, which may be carbon and/or heteroatom(s), with anindependently chosen R^(t) group. For example, Ring T may be selectedfrom but not limited to the following types:

in which n is 0, 1, 2 or 3. It is understood that the total number ofsubstituents R^(t) does not exceed the normal available valencies. Thus,for example, when Ring T is a pyrolle ring, it can optionally besubstituted with 1 to 3 substituents (i.e. n is 0, 1, 2 or 3), whereaswhen Ring T is a pyrazole or an imidazole, it can optionally besubstituted with a maximum of 2 substituents (i.e. n is 0, 1 or 2). Itis also understood that when Ring T is unsubstituted, hydrogen atomsotherwise not depicted are present to meet the desire valency.

Illustrative examples of such compounds include those in which ring Tis:

For the previously described class and subclass of compounds, as in allcompounds of the invention, Ring A and Ring B are as previously definedin part 1.

Illustrative examples of substituted Ring A are:

Ring B represents a 5 or 6-membered aryl or heteroaryl ring as definedabove in part 1. Illustrative examples of substituted Ring B are:

Of special interest is another class of compounds of Formula I asdescribed above in Part 1, in which one of the R^(b) substituents is a5- or 6-membered ring (Ring C), which may be heteroaryl or heterocyclic,comprising of carbon atoms and 1-3 heteroatoms independently selectedfrom O, N and S(O)_(r) and Ring C being optionally substituted on carbonor heteroatom(s) with 1 to 5 substituents R^(c).

This class is represented by compounds of Formula II and a tautomer, anindividual isomer, a mixture of isomers or a pharmaceutically acceptablesalt, solvate or hydrate thereof:

in which the previously defined variables, e.g. n, m, A, B, T, L¹, R¹,R^(t), R^(a) and R^(b) are as defined above in part 1, andR^(c), at each occurrence, is independently selected from halo, ═O, ═S,—CN, —NO₂, —R⁴, —OR², —NR²R³, —C(O)YR², —OC(O)YR², —NR²C(O)YR²,—Si(R⁴)₃, —SC(O)YR², —NR²C(═S)YR², —OC(═S)YR², —C(═S)YR², —YC(═NR)YR²,—YP(═O)(YR⁴)(YR⁴), —NR²SO₂R², —S(O)_(r)R², —SO₂NR²R³ and —NR²SO₂NR²R³,in which Y, r, R², R³ and R⁴ are as defined previously in Part 1; t is0, 1, 2, 3 or 4 and v is 0, 1, 2, 3, 4 or 5.

Illustrative examples of Ring C systems, optionally substituted with 1-5R^(c) groups, include but are not limited to the following types:

in which v and R^(c) are as defined above and in which the total numberof substituents R^(c) does not exceed the normal valencies.

Specific, non-limiting illustrative examples of this class include thefollowing compounds:

in which several illustrative -[Ring A]-[L¹]-[Ring B]-[Ring C] portionsare depicted.

Compounds of interest include among others, compounds of Formula II inwhich Ring C is a heteroaryl ring, unsubstituted or substituted with oneor more R^(c) groups. Of particular current interest, are compounds ofthis subclass in which Ring C is an imidazole ring. Of further interest,are compounds of this subclass in which Ring C bears a single loweralkyl (e.g. methyl) R^(c) group.

A further feature of the invention relates to compounds of Formula I asdescribed in Part 1, in which one of the R^(b) substituents is-[L²]-[Ring D]. This class is represented by Formula III:

in which the variables, e.g.; n, m, Ring T, Ring A, Ring B, L₁, R¹,R^(t), R^(a) and R^(b) are as defined above in part 1,L² is selected from (CH₂)_(z), O(CH₂)_(x), NR³(CH₂)_(x), S(CH₂)_(x), and(CH₂)_(x)NR³C(O)(CH₂)_(x), and the linker moiety L² can be included ineither direction;Ring D represents a 5- or 6-membered heterocyclic or heteroaryl ringcomprising carbon atoms and 1-3 heteroatoms independently selected fromO, N and S(O)_(r), and Ring D is optionally substituted on carbon orheteroatom(s) with 1-5 R^(d) groups;R^(d), at each occurrence, is independently selected from halo, ∇O, ═S,—CN, —NO₂, —R⁴, —OR², —NR²R³, —Si(R⁴)₃, —C(O)YR², —OC(O)YR²,—NR²C(O)YR², —SC(O)YR², —NR²C(═S)YR², —OC(═S)YR², —C(═S)YR²,—YC(═NR³)YR², —YP(═O)(YR²)(YR²), —NR²SO₂R², —S(O)_(r)R², —SO₂NR²R³ and—NR²SO₂NR²R³, wherein Y, r, R², R³ and R⁴ are as previously defined inPart 1;

-   w is 0, 1, 2, 3, 4 or 5;-   x is 0, 1, 2 or 3;-   z is 1, 2, 3 or 4;-   and t is 0, 1, 2, 3 or 4.    Non-limiting, illustrative examples of -[Ring B]-[L²]-[Ring D]    moieties in compounds of Formula III include among others:

Specific, non limiting illustrative examples of this class include thefollowing compounds:

Compounds of interest include among others, compounds of Formula III inwhich Ring D is a heterocyclic ring, such as piperazine ring, optionallysubstituted on the nitrogen with R^(d) and L² is —CH₂. Of particularcurrent interest, are compounds of this subclass in which R^(d) is asubstituted or unsubstituted lower alkyl (i.e., 1-6 carbon alkyl).

Other compounds of interest include among others, compounds of FormulaIII in which Ring D is a heteroaryl ring, unsubstituted or substitutedwith one or more R^(d) groups.

Of special interest for use in this invention are compounds of FormulaII and III, in which Rings A and B are aryl.

Another subclass of interest are compounds of formula II and III, inwhich Ring T is optionally substituted with one or more R^(t) moietiesselected from halo, lower alkyl, alkoxy, amino, —NH-alkyl, —C(O)NH₂,—C(O)O-alkyl, —C(O)NH-alkyl, —NHC(O)-alkyl, —NHC(O)-heterocycle,—NHC(O)NH-alkyl, —NHC(O)NH—(CH₂)_(x)heterocycle, —NHC(NH)-alkyl,—NHC(NH)NH₂, —NHC(NH)O-alkyl, —NH(CH₂)_(x)-heteroaryl,—NH(CH₂)_(x)-heterocycle, —NH(CH₂)_(x)-aryl or —(CH₂)_(x)C(O)NH₂, inwhich x is an integer of 0-3 and alkyl include straight (i.e.,unbranched and acyclic), branched and cyclic alkyl groups, andoptionally substituted and in which aryl, heteroaryl, heterocyclyl ringsare optionally substituted.

Illustrative, non limiting, examples of this subclass include compoundsof formula II and III in which Ring T is one of the following:

A subclass of current interest includes compounds of formula IIa, IIb,IIc IIIa, IIIb, IIIc:

in which the previously defined variables, e.g., R^(a), R^(b), R^(c),R^(d) and R^(t), n, m, and t are as previously defined, e.g., in part 1and in which (R^(d))₀₋₁ represent 0 to 1 R^(d) group. Where Ring D isnot substituted, a hydrogen atom replaces the depicted R^(d) group, tomeet the desired valency as would be obvious to the practitioner. Thenumber of substituents R^(t), R^(c) and R^(d) also do not exceed thenormal valencies.

Of special interest also is a subclass of compounds of formula II andIII, in which Ring T is an imidazole, optionally substituted with one ormore R^(t). Of particular interest, are compounds in which R^(t) isabsent or R^(t) is independently selected from halo, lower alkyl,alkoxy, amino, —NH-alkyl, —C(O)NH₂, —C(O)NH-alkyl, —NHC(O)-alkyl,—NHC(O)NH-alkyl, —NHC(NH)-alkyl, —NHC(O)CH₂N(alkyl)₂, —NHC(NH)NH₂ or(CH₂)C(O)NH₂, in which alkyl include straight (i.e., unbranched oracyclic), branched and cyclic alkyl groups and is optionallysubstituted.

Illustrative, non limiting, examples of this subclass are compounds inwhich substituted Ring T is:

A particular class of interest includes compounds of Formula IIa, IIb,and IIc in which m is 1, t is 1, v is 1, R¹ is independently absent orRt is selected from —C(O)NH₂, —C(O)NHalkyl, NH₂, and lower alkyl, e.g.,—CH₃; R^(a) is lower alkyl, e.g., —CH₃; R^(b) is isopropyl or CF₃ andR^(c) is absent or methyl.

Another subset of interest includes compounds of Formula IIIa, IIIb, andIIIc in which m is 1, t is 1, R^(t) is independently absent or selectedfrom —C(O)NH₂, —C(O)NHalkyl, NH₂, and lower alkyl, e.g., —CH₃; R^(a) islower alkyl, e.g., —CH₃; R^(b) is isopropyl or CF₃and R^(d) is methyl orCH₂CH₂OH.

Compounds of this invention of particular interest include those withone or more of the following characteristics:

a molecular weight of less than 1000, preferably less than 750 and morepreferably less than 600 mass units (not including the weight of anysolvating or co-crystallizing species, of any counter-ion in the case ofa salt); orinhibitory activity against a wild type or mutant (especially aclinically relevant mutant) kinase, especially a Src family kinase suchas Src, Yes, Lyn or Lck; a VEGF-R such as VEGF-R1 (Flt-1), VEGF-R2(kdr), or VEGF-R3; a PDGF-R; an Abl kinase or another kinase of interestwith an IC50 value of 1 μM or less (as determined using anyscientifically acceptable kinase inhibition assay), preferably with anIC50 of 500 nM or better, and optimally with an IC50 value of 250 nM orbetter; orinhibitory activity against a given kinase with an IC50 value at least100-fold lower than their IC50 values for other kinases of interest; orinhibitory activity against both Src and kdr with a 1 μM or better IC50value against each; ora cytotoxic or growth inhibitory effect on cancer cell lines maintainedin vitro, or in animal studies using a scientifically acceptable cancercell xenograft model, (especially preferred are compounds of theinvention which inhibit proliferation of cultured K562 cells with apotency at least as great as Gleevec, preferably with a potency at leasttwice that of Gleevec, and more preferably with a potency at least 10times that of Gleevec as determined by comparative studies.).

Also provided is a composition comprising at least one compound of theinvention or a salt, hydrate or other solvate thereof, and at least onepharmaceutically acceptable excipient or additive. Such compositions canbe administered to a subject in need thereof to inhibit the growth,development and/or metastasis of cancers, including solid tumors (e.g.,breast, colon, pancreatic, CNS and head and neck cancers, among others)and various forms of leukemia, including leukemias and other cancerswhich are resistant to other treatment, including those which areresistant to treatment with Gleevec or another kinase inhibitor, andgenerally for the treatment and prophylaxis of diseases or undesirableconditions mediated by one or more kinases which are inhibited by acompound of this invention.

The cancer treatment method of this invention involves administering (asa monotherapy or in combination with one or more other anti-canceragents, one or more agents for ameliorating side effects, radiation,etc) a therapeutically effective amount of a compound of the inventionto a human or animal in need of it in order to inhibit, slow or reversethe growth, development or spread of cancer, including solid tumors orother forms of cancer such as leukemias, in the recipient. Suchadministration constitutes a method for the treatment or prophylaxis ofdiseases mediated by one or more kinases inhibited by one of thedisclosed compounds or a pharmaceutically acceptable derivative thereof.“Administration” of a compound of this invention encompasses thedelivery to a recipient of a compound of the sort described herein, or aprodrug or other pharmaceutically acceptable derivative thereof, usingany suitable formulation or route of administration, as discussedherein. Typically the compound is administered one or more times permonth, often one or more times per week, e.g. daily, every other day, 5days/week, etc. Oral and intravenous administrations are of particularcurrent interest.

The phrase, “pharmaceutically acceptable derivative”, as used herein,denotes any pharmaceutically acceptable salt, ester, or salt of suchester, of such compound, or any other adduct or derivative which, uponadministration to a patient, is capable of providing (directly orindirectly) a compound as otherwise described herein, or a metabolite orresidue (MW>300) thereof. Pharmaceutically acceptable derivatives thusinclude among others pro-drugs. A pro-drug is a derivative of acompound, usually with significantly reduced pharmacological activity,which contains an additional moiety which is susceptible to removal invivo yielding the parent molecule as the pharmacologically activespecies. An example of a pro-drug is an ester which is cleaved in vivoto yield a compound of interest. Pro-drugs of a variety of compounds,and materials and methods for derivatizing the parent compounds tocreate the pro-drugs, are known and may be adapted to the presentinvention.

Particularly favored derivatives and prodrugs of a parent compound arethose derivatives and prodrugs that increase the bioavailability of thecompound when administered to a mammal (e.g., by permitting enhancedabsorption into the blood following oral administration) or whichenhance delivery to a biological compartment of interest (e.g., thebrain or lymphatic system) relative to the parent compound. Preferredprodrugs include derivatives of a compound of this invention withenhanced aqueous solubility or active transport through the gutmembrane, relative to the parent compound.

One important aspect of this invention is a method for treating cancerin a subject in need thereof, which comprises administering to thesubject a treatment effective amount of a composition containing acompound of this invention. Various cancers which may be thus treatedare noted elsewhere herein and include, among others, cancers which areor have become resistant to another anticancer agent such as Gleevec,Iressa, Tarceva or one of the other agents noted herein. Treatment maybe provided in combination with one or more other cancer therapies,include surgery, radiotherapy (e.g., gamma-radiation, neutron beamradiotherapy, electron beam radiotherapy, proton therapy, brachytherapy,and systemic radioactive isotopes, etc.), endocrine therapy, biologicresponse modifiers (e.g., interferons, interleukins, and tumor necrosisfactor (TNF) to name a few), hyperthermia, cryotherapy, agents toattenuate any adverse effects (e.g., antiemetics), and other cancerchemotherapeutic drugs. The other agent(s) may be administered using aformulation, route of administration and dosing schedule the same ordifferent from that used with the compound of this invention.

Such other drugs include but not limited to one or more of thefollowing: an anti-cancer alkylating or intercalating agent (e.g.,mechlorethamine, chlorambucil, Cyclophosphamide, Melphalan, andIfosfamide); antimetabolite (e.g., Methotrexate) purine antagonist orpyrimidine antagonist (e.g., 6-Mercaptopurine, 5-Fluorouracil,Cytarabile, and Gemcitabine); spindle poison (e.g., Vinblastine,Vincristine, Vinorelbine and Paclitaxel); podophyllotoxin (e.g.,Etoposide, Irinotecan, Topotecan); antibiotic (e.g., Doxorubicin,Bleomycin and Mitomycin); nitrosourea (e.g., Carmustine, Lomustine);inorganic ion (e.g., Cisplatin, Carboplatin, Oxaliplatin or oxiplatin);enzyme (e.g., Asparaginase); hormone (e.g., Tamoxifen, Leuprolide,Flutamide and Megestrol); mTOR inhibitor (e.g., Sirolimus (rapamycin),Temsirolimus (CCI779), Everolimus (RAD001), AP23573 or other compoundsdisclosed in U.S. Pat. No. 7,091,213); proteasome inhibitor (such asVelcade, another proteasome inhibitor (see e.g., WO 02/096933) oranother NF-kB inhibitor, including, e.g., an IkK Inhibitor); otherkinase inhibitors (e.g., an inhibitor of Src, BRC/Abl, kdr, flt3,aurora-2, glycogen synthase kinase 3 (“GSK-3”), EGF-R kinase (e.g.,Iressa, Tarceva, etc.), VEGF-R kinase, PDGF-R kinase, etc); an antibody,soluble receptor or other receptor antagonist against a receptor orhormone implicated in a cancer (including receptors such as EGFR, ErbB2,VEGFR, PDGFR, and IGF-R; and agents such as Herceptin, Avastin, Erbitux,etc.); etc. For a more comprehensive discussion of updated cancertherapies see, http://www.nci.nih.gov/, a list of the FDA approvedoncology drugs at http://www.fda.gov/cder/cancer/druglistframe.htm, andThe Merck Manual, Seventeenth Ed. 1999, the entire contents of which arehereby incorporated by reference. Examples of other therapeutic agentsare noted elsewhere herein and include among others, Zyloprim,alemtuzmab, altretamine, amifostine, nastrozole, antibodies againstprostate-specific membrane antigen (such as MLN-591, MLN591RL andMLN2704), arsenic trioxide, bexarotene, bleomycin, busulfan,capecitabine, Gliadel Wafer, celecoxib, chlorambucil,cisplatin-epinephrine gel, cladribine, cytarabine liposomal,daunorubicin liposomal, daunorubicin, daunomycin, dexrazoxane,docetaxel, doxorubicin, Elliott's B Solution, epirubicin, estramustine,etoposide phosphate, etoposide, exemestane, fludarabine, 5-FU,fulvestrant, gemcitabine, gemtuzumab-ozogamicin, goserelin acetate,hydroxyurea, idarubicin, idarubicin, idamycin, ifosfamide, imatinibmesylate, irinotecan (or other topoisomerase inhibitor, includingantibodies such as MLN576 (XR11576)), letrozole, leucovorin, leucovorinlevamisole, liposomal daunorubicin, melphalan, L-PAM, mesna,methotrexate, methoxsalen, mitomycin C, mitoxantrone, MLN518 or MLN608(or other inhibitors of the flt-3 receptor tyrosine kinase, PDFG-R orc-kit), itoxantrone, paclitaxel, Pegademase, pentostatin, porfimersodium, Rituximab (RITUXAN®), talc, tamoxifen, temozolamide, teniposide,VM-26, topotecan, toremifene, 2C4 (or other antibody which interfereswith HER2-mediated signaling), tretinoin, ATRA, valrubicin, vinorelbine,or pamidronate, zoledronate or another bisphosphonate.

This invention further comprises the preparation of a compound of any ofFormula I, II, III, IIa, IIb, IIc, IIIa, IIIb, IIIc or of any other ofthe compounds of this invention.

The invention also comprises the use of a compound of the invention, ora pharmaceutically acceptable derivative thereof, in the manufacture ofa medicament for the treatment either acutely or chronically of cancer(including leukemias and solid tumors, primary or metastatic, includingcancers such as noted elsewhere herein and including cancers which areresistant or refractory to one or more other therapies). The compoundsof this invention are useful in the manufacture of an anti-cancermedicament. The compounds of the present invention are also useful inthe manufacture of a medicament to attenuate or prevent disordersthrough inhibition of one or more kinases such as Src, kdr, abl. etc.

Other disorders which may be treated with a compound of this inventioninclude metabolic disorders, inflammatory disorders and osteoporosis andother bone disorders. In such cases the compound of this invention maybe used as a monotherapy or may be administered in conjunction withadministration of another drug for the disorder, e.g., a bisphosphonatein the case of osteoporosis or other bone-related illnesses.

This invention further encompasses a composition comprising a compoundof the invention, including a compound of any of the described classesor subclasses, including those of any of the formulas noted above, amongothers, preferably in a therapeutically-effective amount, in associationwith a least one pharmaceutically acceptable carrier, adjuvant ordiluent.

Compounds of this invention are also useful as standards and reagentsfor characterizing various kinases, especially but not limited to kdrand Src family kinases, as well as for studying the role of such kinasesin biological and pathological phenomena; for studying intracellularsignal transduction pathways mediated by such kinases, for thecomparative evaluation of new kinase inhibitors; and for studyingvarious cancers in cell lines and animal models.

3. Definitions

In reading this document, the following information and definitionsapply unless otherwise indicated. In addition, unless otherwiseindicated, all occurrences of a functional group are independentlychosen, as the reader is in some cases reminded by the use of a slashmark or prime to indicate simply that the two occurrences may be thesame or different (e.g., R, R′, R″, or Y, Y′, Y″ etc.).

The term “Alkyl” is intended to include linear (i.e., unbranched oracyclic), branched, cyclic, or polycyclic non aromatic hydrocarbongroups, which are optionally substituted with one or more functionalgroups. Unless otherwise specified, “'alkyl” groups contain one toeight, and preferably one to six carbon atoms. C₁₋₆ alkyl, is intendedto include C₁, C₂, C₃, C₄, C₅, and C₆ alkyl groups. Lower alkyl refersto alkyl groups containing 1 to 6 carbon atoms. Examples of Alkylinclude, but are not limited to, methyl, ethyl, n-propyl, isopropyl,cyclopropyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, pentyl,isopentyl tert-pentyl, cyclopentyl, hexyl, isohexyl, cyclohexyl, etc.Alkyl may be substituted or unsubstituted. Illustrative substitutedalkyl groups include, but are not limited to, fluoromethyl,difluoromethyl, trifluoromethyl, 2-fluoroethyl, 3-fluoropropyl,hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, benzyl, substitutedbenzyl, phenethyl, substituted phenethyl, etc.

The term “Alkoxy” represents a subset of alkyl in which an alkyl groupas defined above with the indicated number of carbons attached throughan oxygen bridge. For example, “alkoxy” refers to groups —O-alkyl,wherein the alkyl group contains 1 to 8 carbons atoms of a linear,branched, cyclic configuration. Examples of “alkoxy” include, but arenot limited to, methoxy, ethoxy, n-propoxy, l-propoxy, t-butoxy,n-butoxy, s-pentoxy and the like.

“Haloalkyl” is intended to include both branched and linear chainsaturated hydrocarbon having one or more carbon substituted with aHalogen. Examples of haloalkyl, include, but are not limited to,trifluoromethyl, trichloromethyl, pentafluoroethyl and the like.

The term “alkenyl” is intended to include hydrocarbon chains of linear,branched, or cyclic configuration having one or more unsaturatedCarbon-carbon bonds that may occur in any stable point along the chainor cycle. Unless otherwise specified, “alkenyl” refers to groups usuallyhaving two to eight, often two to six carbon atoms. For example,“alkenyl” may refer to prop-2-enyl, but-2-enyl, but-3-enyl,2-methylprop-2-enyl, hex-2-enyl, hex-5-enyl, 2,3-dimethylbut-2-enyl, andthe like. Furthermore, alkenyl groups may be substituted orunsubstituted.

The term “alkynyl” is intended to include hydrocarbon chains of eitherlinear or branched configuration, having one or more carbon-carbontriple bond that may occur in any stable point along the chain. Unlessotherwise specified, “alkynyl” groups refer refers to groups having twoto eight, preferably two to six carbons. Examples of “alkynyl” include,but are not limited to prop-2-ynyl, but-2-ynyl, but-3-ynyl, pent-2-ynyl,3-methylpent-4-ynyl, hex-2-ynyl, hex-5-ynyl, etc. Furthermore, alkynylgroups may be substituted or unsubstituted.

Cycloalkyl is a subset of alkyl and includes any stable cyclic orpolycyclic hydrocarbon groups of from 3 to 13 carbon atoms, any of whichis saturated. Examples of such cycloalkyl include, but are not limitedto cyclopropyl, norbornyl, [2.2.2]bicyclooctane, [4.4.0]bicyclodecane,and the like, which, as in the case of other alkyl moieties, mayoptionally be substituted. The term “cycloalkyl” may be usedinterchangeably with the term “carbocycle”.

Cycloalkenyl is a subset of alkenyl and includes any stable cyclic orpolycyclic hydrocarbon groups of from 3 to 13 carbon atoms, preferablyfrom 5 to 8 carbon atoms, which contains one or more unsaturatedcarbon-carbon double bonds that may occur in any point along the cycle.Examples of such cycloalkenyl include, but are not limited tocyclopentenyl, cyclohexenyl and the like.

Cycloalkynyl is a subset of alkynyl and includes any stable cyclic orpolycyclic hydrocarbon groups of from 5 to 13 carbon atoms, whichcontains one or more unsaturated carbon-carbon triple bonds that mayoccur in any point along the cycle. As in the case of other alkenyl andalkynyl moieties, cycloalkenyl and cycloalkynyl may optionally besubstituted.

“Heterocycle”, “heterocyclyl”, or “heterocyclic” as used herein refersto non-aromatic ring systems having five to fourteen ring atoms,preferably five to ten, in which one or more ring carbons, preferablyone to four, are each replaced by a heteroatom such as N, O, or S.Non-limiting examples of heterocyclic rings include3-1H-benzimidazol-2-one, (1-substituted)-2-oxo-benzimidazol-3-yl,2-tetrahydrofuranyl, 3-tetrahydrofuranyl, 2-tetrahydrothiophenyl,3-tetrahydrothiophenyl, 2-morpholinyl, 3-morpholinyl, 4-morpholinyl,2-thiomorpholinyl, 3-thiomorpholinyl, 4-thiomorpholinyl, 1-pyrrolidinyl,2-pyrrolidinyl, 3-pyrrolidinyl, 1-piperazinyl, 2-piperazinyl,1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl,4-thiazolidinyl, diazolonyl, N-substituted diazolonyl, 1-phthalimidinyl,benzoxanyl, benzopyrrolidinyl, benzopiperidinyl, benzoxolanyl,benzothiolanyl, and benzothianyl. Also included within the scope of theterm “heterocyclyl” or “heterocyclic”, as it is used herein, is a groupin which a non-aromatic heteroatom-containing ring is fused to one ormore aromatic or non-aromatic rings, such as in an indolinyl, chromanyl,phenanthridinyl, or tetrahydroquinolinyl, where the radical or point ofattachment is on the non-aromatic heteroatom-containing ring. The term“heterocycle”, “heterocyclyl”, or “heterocyclic” whether saturated orpartially unsaturated, also refers to rings that are optionallysubstituted.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to aromatic ring groupshaving six to fourteen ring atoms, such as phenyl, 1-naphthyl,2-naphthyl, 1-anthracyl and 2-anthracyl. An “aryl” ring may contain oneor more substituents. The term “aryl” may be used interchangeably withthe term “aryl ring”. “Aryl” also includes fused polycyclic aromaticring systems in which an aromatic ring is fused to one or more rings.Non-limiting examples of useful aryl ring groups include phenyl,hydroxyphenyl, halophenyl, alkoxyphenyl, dialkoxyphenyl,trialkoxyphenyl, alkylenedioxyphenyl, naphthyl, phenanthryl, anthryl,phenanthro and the like, as well as 1-naphthyl, 2-naphthyl, 1-anthracyland 2-anthracyl. Also included within the scope of the term “aryl”, asit is used herein, is a group in which an aromatic ring is fused to oneor more non-aromatic rings, such as in a indanyl, phenanthridinyl, ortetrahydronaphthyl, where the radical or point of attachment is on thearomatic ring.

The term “heteroaryl” as used herein refers to stable heterocyclic, andpolyheterocyclic aromatic moieties having 5-14 ring atoms. Heteroarylgroups may be substituted or unsubstituted and may comprise one or morerings. Examples of typical heteroaryl rings include 5-memberedmonocyclic ring groups such as thienyl, pyrrolyl, imidazolyl, pyrazolyl,furyl, isothiazolyl, furazanyl, isoxazolyl, thiazolyl and the like;6-membered monocyclic groups such as pyridyl, pyrazinyl, pyrimidinyl,pyridazinyl, triazinyl and the like; and polycyclic heterocyclic ringgroups such as benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl,isobenzofuranyl, chromenyl, xanthenyl, phenoxathienyl, indolizinyl,isoindolyl, indolyl, indazolyl, purinyl, isoquinolyl, quinolyl,phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, benzothiazole,benzimidazole, tetrahydroquinoline cinnolinyl, pteridinyl, carbazolyl,beta-carbolinyl, phenanthridinyl, acridinyl, perimidinyl,phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl, phenoxazinyl,and the like (see e.g. Katritzky, Handbook of Heterocyclic Chemistry).Further specific examples of heteroaryl rings include 2-furanyl,3-furanyl, N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl,3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-oxadiazolyl, 5-oxadiazolyl,2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-pyrimidyl,3-pyridazinyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 5-tetrazolyl,2-triazolyl, 5-triazolyl, 2-thienyl, 3-thienyl, carbazolyl,benzimidazolyl, benzothienyl, benzofuranyl, indolyl, quinolinyl,benzotriazolyl, benzothiazolyl, benzooxazolyl, benzimidazolyl,isoquinolinyl, indolyl, isoindolyl, acridinyl, or benzoisoxazolyl.Heteroaryl groups further include a group in which a heteroaromatic ringis fused to one or more aromatic or nonaromatic rings where the radicalor point of attachment is on the heteroaromatic ring. Examples includetetrahydroquinoline, tetrahydroisoquinoline, andpyrido[3,4-d]pyrimidinyl, imidazo[1,2-a]pyrimidyl,imidazo[1,2-a]pyrazinyl, imidazo[1,2-a]pyiridinyl,imidazo[1,2-c]pyrimidyl, pyrazolo[1,5-a][1,3,5]triazinyl,pyrazolo[1,5-c]pyrimidyl, imidazo[1,2-b]pyridazinyl,imidazo[1,5-a]pyrimidyl, pyrazolo[1,5-b][1,2,4]triazine, quinolyl,isoquinolyl, quinoxalyl, imidazotriazinyl, pyrrolo[2,3-d]pyrimidyl,triazolopyrimidyl, pyridopyrazinyl. The term “heteroaryl” also refers torings that are optionally substituted. The term “heteroaryl” may be usedinterchangeably with the term “heteroaryl ring” or the term“heteroaromatic”.

An aryl group (including the aryl portion of an aralkyl, aralkoxy, oraryloxyalkyl moiety and the like) or heteroaryl group (including theheteroaryl portion of a heteroaralkyl or heteroarylalkoxy moiety and thelike) may contain one or more substituents. Examples of suitablesubstituents on the unsaturated carbon atom of an aryl or heteroarylgroup include halogen (F, Cl, Br or I), alkyl, alkenyl, alkynyl, —CN,—R⁴, —OR², —S(O)_(r)R², (wherein r is an integer of 0, 1 or 2),—SO₂NR²R³, —NR²R³, —(CO)YR², —O(CO)YR², —NR²(CO)YR², —S(CO)YR²,—NR²C(═S)YR², —OC(═S)YR², —C(═S)YR², wherein each occurrence of Y isindependently —O—, —S—, —NR³—, or a chemical bond; —(CO)YR² thusencompasses —C(═O)R², —C(═O)OR², and —C(═O)NR²R³. Additionalsubstituents include —YC(═NR³)YR², —COCOR², —COMCOR² (where M is a 1-6carbon alkyl group), —YP(═O)(YR⁴)(YR⁴) (including among others—P(═O)(R⁴)₂), —Si(R⁴)₃, —NO₂, —NR²SO₂R² and —NR²SO₂NR²R³. To illustratefurther, substituents in which Y is —NR³ thus include among others,—NR³C(═O)R², —NR³C(═O)NR²R³, —NR³C(═O)OR², and —NR³C(=NH)NR²R³. R⁴substituent is selected from alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heterocyclyl; R² and R³substituents at each occurrence are independently selected fromhydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, aryl, heteroaryl, heterocyclyl, and R², R³ and R⁴substituents may themselves be substituted or unsubstituted. Examples ofsubstituents allowed on R², R³ and R⁴ include, among others amino,alkylamino, dialkylamino, aminocarbonyl, halogen, alkyl, aryl,heteroaryl, carbocycle, heterocycle, alkylaminocarbonyl,dialkylaminocarbonyl, alkylaminocarbonyloxy, dialkylaminocarbonyloxy,nitro, cyano, carboxy, alkoxycarbonyl, alkylcarbonyl, hydroxy, alkoxy,haloalkoxy groups. Additional illustrative examples include protected OH(such as acyloxy), phenyl, substituted phenyl, —O-phenyl,—O-(substituted) phenyl, -benzyl, substituted benzyl, —O-phenethyl(i.e., —OCH₂CH₂C₆H₅), —O-(substituted)phenethyl. Non-limitingillustrations of a substituted R², R³ or R⁴ moiety include haloalkyl andtrihaloalkyl, alkoxyalkyl, halophenyl, -M-heteroaryl, -M-heterocycle,-M-aryl, -M-OR², -M-SR² -M-NR²R³, -M-OC(O)NR²R³, -M-C(═NR²)NR²R³,-M-C(═NR²)OR³, -M-P(O)R²R³, Si(R²)₃, -M-NR²C(O)R³, -M-NR²C(O)OR²,-M-C(O)R², -M-C(═S)R², -M-C(═S)NR²R³, -M-C(O)NR²R³, -M-C(O)NR²-M-NR²R³,-M-NR²C(NR³)NR²R³, -M-NR²C(S)NR²R³, -M-S(O)₂R³, -M-C(O)R³, -M-OC(O)R³,-MC(O)SR², -M-S(O)₂NR²R³, —C(O)-M-C(O)R², -MCO₂R², -MC(═O)NR²R³,-M-C(═NH)NR²R³, and -M-OC(═NH)NR²R³ (wherein M is a 1-6 carbon alkylgroup).

Some more specific examples include but are not limited to chloromethyl,trichloromethyl, trifluoromethyl, methoxyethyl, alkoxyphenyl,halophenyl, —CH₂-aryl, —CH₂-heterocycle, —CH₂C(O)NH₂, —C(O)CH₂N(CH₃)₂,—CH₂CH₂OH, —CH₂OC(O)NH₂, —CH₂CH₂NH₂, —CH₂CH₂CH₂NEt₂, —CH₂OCH₃, —C(O)NH₂,—CH₂CH₂-heterocycle, —C(═S)CH₃, —C(═S)NH₂, —C(═NH)NH₂, —C(═NH)OEt,—C(O)NH-cyclopropyl, C(O)NHCH₂CH₂-heterocycle, —C(O)NHCH₂CH₂OCH₃,—C(O)CH₂CH₂NHCH₃, —CH₂CH₂F, —C(O)CH₂-heterocycle, —CH₂C(O)NHCH₃,—CH₂CH₂P(O)(CH₃)₂, Si(CH₃)₃ and the like.

An alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, cycloalkyl, cycloalkenyl,cycloalkynyl or non-aromatic heterocyclic group may thus also containone or more substituents. Examples of suitable substituents on suchgroups include, but are not limited to those listed above for the carbonatoms of an aryl or heteroaryl group and in addition include thefollowing substituents for a saturated carbon atom: ═O, ═S, ═NH,═NNR²R³, ═NNHC(O)R², ═NNHCO₂R², or ═NNHSO₂R², wherein R² and R³ at eachoccurrence are independently hydrogen, alkyl, alkenyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heterocyclyl.Illustrative examples of substituents on an aliphatic, heteroaliphaticor heterocyclic group include amino, alkylamino, dialkylamino,aminocarbonyl, halogen, alkyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylaminocarbonyloxy, dialkylaminocarbonyloxy, alkoxy, nitro, —CN,carboxy, alkoxycarbonyl, alkylcarbonyl, —OH, haloalkoxy, or haloalkylgroups.

Illustrative substituents on a nitrogen, e.g., in an heteroaryl ornon-aromatic heterocyclic ring include R⁴, —NR²R³, —C(═O)R², —C(═O)OR²,—C(=O)SR², —C(═O)NR²R³, —C(═NR²)NR²R³, —C(═NR²)OR², —C(═NR²)R³, —COCOR²,—COMCOR², —CN, —SO₂R³, S(O)R³, —P(═O)(YR²)(YR²), —NR²SO₂R³ and—NR²SO₂NR²R³, wherein each occurrence of R⁴ is alkyl, alkenyl, alkynyl,cycloalkkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl andheterocyclyl; each occurrence of R² and R³ is independently hydrogen,alkyl, alkenyl, alkynyl, cycloalkkyl, cycloalkenyl, cycloalkynyl, aryl,heteroaryl and heterocyclyl.

When a ring system (e.g., cycloalkyl, heterocyclyl, aryl, or heteroaryl)is substituted with a number of substituents varying within an expresslydefined range, it is understood that the total number of substituentsdoes not exceed the normal available valencies under the existingconditions. Thus, for example, a phenyl ring substituted with “p”substituents (where “p” ranges from 0 to 5) can have 0 to 5substituents, whereas it is understood that a pyridinyl ring substitutedwith “p” substituents has a number of substituents ranging from 0 to 4.The maximum number of substituents that a group in the compounds of theinvention may have can be easily determined.

This invention encompasses only those combinations of substituents andvariables that result in a stable or chemically feasible compound. Astable compound or chemically feasible compound is one that hasstability sufficient to permit its preparation and detection. Preferredcompounds of this invention are sufficiently stable that they are notsubstantially altered when kept at a temperature of 40° C. or less, inthe absence of moisture or other chemically reactive conditions, for atleast a week.

Certain compounds of this invention may exist in tautomeric forms, andthis invention includes all such tautomeric forms of those compoundsunless otherwise specified.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Thus, single stereochemicalisomers as well as enantiomeric and diastereomeric mixtures of thepresent compounds are within the scope of the invention. Thus, thisinvention encompasses each diasteriomer or enantiomer substantially freeof other isomers (>90%, and preferably >95%, free from otherstereoisomers on a molar basis) as well as a mixture of such isomers.

Particular optical isomers can be obtained by resolution of the racemicmixtures according to conventional processes, e.g., by formation ofdiastereoisomeric salts, by treatment with an optically active acid orbase. Examples of appropriate acids are tartaric. diacetyltartaric,dibenzoyltartaric, ditoluoyltartaric, and camphorsulfonic acid and thenseparation of the mixture of diastereoisomers by crystallizationfollowed by liberation of the optically active bases from these salts. Adifferent process for separation of optical isomers involves the use ofa chiral chromatography column optimally chosen to maximize theseparation of the enantiomers. Still another method involves synthesisof covalent diastereoisomeric molecules by reacting compounds of theinvention with an optically pure acid in an activated form or anoptically pure isocyanate. The synthesized diastereoisomers can beseparated by conventional means such as chromatography, distillation,crystallization or sublimation, and then hydrolyzed to deliver theenantiomerically pure compound.

Optically active compounds of the invention can be obtained by usingactive starting materials. These isomers may be in the form of a freeacid, a free base, an ester or a salt.

The compounds of this invention can exist in radiolabelled form, i.e.,said compounds may contain one or more atoms containing an atomic massor mass number different from the atomic mass or mass number: ordinarilyfound in nature. Radioisotopes of hydrogen, carbon, phosphorous,fluorine and chlorine include ³H, ¹⁴C, ³²P, ³⁵S, ⁴³F and ³⁶Cl,respectively. Compounds of this invention which contain thoseradioisotopes and/or other radioisotopes of other atoms are within thescope of this invention. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C,radioisotopes are particularly preferred for their ease of preparationand detectability.

Radiolabelled compounds of this invention can generally be prepared bymethods well known to those skilled in the art. Conveniently, suchradiolabelled compounds can be prepared by carrying out the proceduresdisclosed herein except substituting a readily available radiolabelledreagent for a non-radiolabelled reagent.

4. Synthetic Overview

The practitioner has a well-established literature of heterocyclic andother relevant chemical transformations, recovery and purificationtechnologies to draw upon, in combination with the information containedin the examples which follow, for guidance on synthetic strategies,protecting groups, and other materials and methods useful for thesynthesis, recovery and characterization of the compounds of thisinvention, including compounds containing the various choices for theR^(t), R^(a), R^(b), R^(c), R^(d), R^(e) and Rings T, A, B, C and D.

Various synthetic approaches may be used to produce the compoundsdescribed herein, including those approaches depicted schematicallybelow. The practitioner will appreciate that protecting groups may beused in these approaches. “Protecting groups”, are moieties that areused to temporarily block chemical reaction at a potentially reactivesite (e.g., an amine, hydroxy, thiol, aldehyde, etc.) so that a reactioncan be carried out selectively at another site in a multifunctionalcompound. In preferred embodiments, a protecting group reactsselectively in good yield to give a protected substrate that is suitablefor the planned reactions; the protecting group should be selectivelyremovable in good yield by readily available, preferably nontoxicreagents that do not unduly attack the other functional groups present;the protecting group preferably forms an readily separable derivative(more preferably without the generation of new stereogenic centers); andthe protecting group preferably has a minimum of additionalfunctionality to avoid the complication of further sites of reaction. Awide variety of protecting groups and strategies, reagents andconditions for deploying and removing them are known in the art. See,e.g., “Protective Groups in Organic Synthesis” Third Ed. Greene, T. W.and Wuts, P. G., Eds., John Wiley & Sons, New York: 1999. For additionalbackground information on protecting group methodologies (materials,methods and strategies for protection and deprotection) and othersynthetic chemistry transformations useful in producing the compoundsdescribed herein, see in R. Larock, Comprehensive organicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 3rd. Ed., John Wiley and Sons(1999); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995). The entire contents of these references are hereby incorporatedby reference.

Also, one may chose reagents enriched for a desired isotope, e.g.deuterium in place of hydrogen, to create compounds of this inventioncontaining such isotope(s). Compounds containing deuterium in place ofhydrogen in one or more locations, or containing various isotopes of C,N, P and O, are encompassed by this invention and may be used, forinstance, for studying metabolism and/or tissue distribution of thecompounds or to alter the rate or path of metabolism or other aspects ofbiological functioning.

The compounds of this invention can be synthesized using the methodsdescribed below, together with synthetic methods known in the art ofsynthetic organic chemistry, or by a variation thereon as appreciated bythose skilled in the art. Preferred methods include, but are not limitedto those described below. The reactions are preformed in a solventappropriate to the reagents and materials employed and suitable for thetransformation being effected. It will be understood by those skilled inthe art of organic synthesis that the functionality present on themolecule should be consistent the transformations proposed. This willsometimes required some judgment to modify the order of the syntheticsteps or to select one particular process scheme over another in orderto obtain a desired compound of the invention.

A compound of the present invention could be prepared as outlined inScheme I to Scheme XVII and via standard methods known to those skilledin the art.

A palladium catalyzed Sonogashira coupling reaction is used to link the‘top’ Ring to the ‘bottom’ [RingA]-[L¹]-[RingB] moiety as illustrated inScheme I, and II. In Scheme I, the Sonogashira coupling reaction isperformed with an acetylenic “top” Ring T and a [RingA]-[L¹]-[RingB]moiety which has been activated by the presence of a reactive group W,which is an iodide, a bromide or another reactive group permitting thedesired coupling reaction. The variables in the intermediateW-[RingA]-[L¹]-[RingB] are as defined previously, Rings A and B beingoptionally substituted with permitted R^(a) and R^(b) groupsrespectively.

An alternative coupling reaction is described in Scheme II, in which theRing T is “activated” by the presence of a reactive group W (such asiodide or bromide) and is coupled to the ‘bottom’ acetylenic[RingA]-[L^(1])-[RingB] under similar Palladium catalyzed couplingconditions.

The Sonogashira coupling conditions described in Scheme I and II areapplicable to all monocyclic heteroaryl Ring T's of this invention andcan be used to synthesize all compounds of this invention.

Several illustrative overall synthetic approaches to the preparation ofthe acetylenic Ring T moieties, based on known transformations, areillustrated below in Scheme III to Scheme VI:

As one of ordinary skill in the art would recognize, these methods forthe preparation of various substituted acetylenic Ring T groups arewidely applicable to various other monocyclic heteraoaryl rings notshown.

Schemes VII to XI below depict the synthesis of compounds of formulaW-[RingA]-[L¹]-[RingB] which are useful intermediates in the couplingreaction described in Schemes I and II.

It should be apparent that intermediates of the formula:

are of particular interest as their coupling reaction with the ‘top’heteroaryl rings produces compounds of the present invention. Thevariable groups A, and B are as previously defined and are optionallysubstituted as described herein, and W is I or an alternative reactivegroup permitting the desired coupling reaction.Illustrative such intermediates include among others those of thosefollowing structures:

wherein the variables. e.g., R^(a), R^(b), R^(c) and R^(d) groups are aspreviously defined. For instance, R^(a) in some embodiments is chosenfrom F or alkyl, e.g., Me, among others, and R^(b) in some embodimentsis chosen from Cl, F, Me, t-butyl, —CF₃ or —OCF₃ among others. Those andother compounds of the formula W-[Ring A]-[L¹]-[Ring B] with the variouspermitted substituents are useful for preparing the correspondingcompounds of the invention as are defined in the various formulae,classes and subclasses disclosed herein.

Some illustrative synthetic routes for the preparation of reagents andrepresentative intermediates are presented below:

Scheme VII describes an illustrative synthesis of the intermediatesW-[RingA]-[L¹]-[RingB] in which Rings A and B are phenyl and L¹ isNHC(O).

Scheme VIII depicts a variant of the foregoing in which Ring B is a2-pyridine and L¹ is C(O)NH (i.e., in the other direction).

Schemes IX and X, below illustrate the synthesis ofW-[RingA]-[L¹]-[RingB] in which Rings A and B are phenyl and ring C isan heteroaryl ring. These intermediates are useful for making compoundsof formula II.

More specifically, scheme IX describes the preparation of intermediatesin which Ring C is an imidazole ring.

Scheme X describes the preparation of intermediates in which Ring C is apyrrole or an oxazole ring.

Scheme XI illustrates the synthesis of W-[RingA]-[L¹]-[RingB] in whichRings A and B are phenyl and an R^(b) substituent is -[L²]-[Ring D].These intermediates are useful for making compounds of formula III inwhich Ring D is a 5 or 6-membered heterocycle, containing one or twoheteroatoms.

In this scheme, non limiting examples of substituents R^(b) on Ring Bare halo, e.g., Cl; lower alkyl groups, e.g., isopropyl; and substitutedlower alkyl groups, e.g., —CF₃; and non limiting examples of Ring D areN,N-dimethylpyrrolidine, N-(2-hydroxyethyl)piperazine, andN-methylpiperazine.

Intermediates W-[Ring A]-[L¹]-[Ring B], such as those presented in thevarious synthetic schemes above, can be reacted with an acetylenic RingT using the Sonogashira coupling conditions described in the generalScheme I.

An example is depicted below in Scheme XII, in which Ring T moiety canbe further derivatized after the Sonogashira coupling step, to generatevarious interesting substituted analogs of this invention.

Alternatively, the intermediates W-[Ring A]-[L¹]-[Ring B] can be reactedunder Sonogashira conditions with trimethylsilylacetylene, prior to thecoupling with an iodo- or a bromo-activated Ring T as otherwisedescribed in the general Scheme II.

An example is depicted in Scheme XIII.

In other embodiments, the steps can be carried out in a different order.For example, the Sonogashira Coupling reaction can be used to connectRing T to Ring A prior to linking that portion to Ring B and/or [RingB]-[L²]-[Ring D] and/or [Ring B]-[Ring C] as shown in Scheme XIV.

In a non-limiting example in which Ring A and Ring B are phenyl and L¹is CONH, Scheme XV describes Sonogashira Coupling of an acetylenic RingT with 3-iodo-4-methylbenzoic acid (a Ring A moiety) to generate a [RingT]-[Ring A] intermediate which then undergoes an amide coupling with anoptionally substituted Ring B moiety:

This approach is illustrated in Scheme XVI which depicts the coupling ofan acetylenic Ring T (i.e.,5-ethynyl-1-methyl-1H-imidazole-2-carboxamide) with a substitutedW-[Ring A] (i.e., 3-iodo-4-methylbenzoic acid), followed by an amidecoupling of the resultant [Ring T]-[Ring A]-COOH intermediate with aH₂N-[Ring B]-L2-[Ring C] moiety (i.e.,4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethylaniline):

Alternatively, as another illustration of the practitioner's range ofassembly options, the 3-iodo-4-methylbenzoic acid Ring A intermediatecan be reacted in a Sonogashira reaction with trimethylsilylacetylene,which after silyl deprotection, can a second Sonogashira couplingreaction with an activated Ring T as illustrated in Scheme XVII.

With synthetic approaches such as the foregoing, combined with theexamples which follow, additional information provided herein andconventional methods and materials, the practitioner should be able toprepare the full range of compounds disclosed herein.

5. Uses, Formulations, Administration Pharmaceutical Uses; Indications

This invention provides compounds having biological properties whichmake them of interest for treating or ameliorating diseases in whichkinases may be involved, symptoms of such disease, or the effect ofother physiological events mediated by kinases. For instance, a numberof compounds of this invention have been shown to inhibit tyrosinekinase activity of Src and abl, among other tyrosine kinases which arebelieved to mediate the growth, development and/or metastasis of cancer.A number of compounds of the invention have also been found to possesspotent in vitro activity against cancer cell lines, including amongothers K-562 leukemia cells. Observed potencies have been as much as10-fold more powerful than Gleevec in conventional antiproliferationassays with K562 cells.

Such compounds are thus of interest for the treatment of cancers,including both primary and metastatic cancers, including solid tumors aswell as lymphomas and leukemias (including CML, AML and ALL), andincluding cancers which are resistant to other therapies, includingother therapies involving the administration of kinase inhibitors suchas Gleevec, Tarceva or Iressa.

Such cancers include, among others, cancers of the breast, cervix, colonand rectum, lung, ovaries, pancreas, prostate, head and neck,gastrointestinal stroma, as well as diseases such as melanoma, multiplemyeloma, non-Hodgkin's lymphoma, melanoma, gastric cancers and leukemias(e.g., myeloid, lymphocytic, myelocytic and lymphoblastic leukemias)including cases which are resistant to one or more other therapies,including among others, Gleevec, Tarceva or Iressa.

Resistance to various anticancer agents can arise from one or moremutations in a mediator or effector of the cancer (e.g., mutation in akinase such as Src or Abl) which correlate with alteration in theprotein's drug binding properties, phosphate binding properties, proteinbinding properties, autoregulation or other characteristics. Forexample, in the case of BCR-Abl, the kinase associated with chronicmyeloid leukemia, resistance to Gleevec has been mapped to a variety ofBCR/Abl mutations which are linked to a variety of functionalconsequences, including among others, steric hindrance of drug occupancyat the kinase's active site, alteration in deformability of thephosphate binding P loop, effects on the conformation of the activationloop surrounding the active site, and others. See e.g. Shah et al, 2002,Cancer Cell 2, 117-125 and Azam et al, 2003, Cell 112, 831-843 andreferences cited therein for representative examples of such mutationsin Bcr/Abl which correlate with drug resistance. See also the followingreferences for additional background information on BCR/Abl, itsmechanistic role in CML and drug-resistance-conferring mechanisms andmutations: Kurzrock et al., Philadelphia chromosome-positive leukemias:from basic mechanisms to molecular therapeutics, Ann Intern Med. 2003May 20; 138(10):819-30; O'Dwyer et al., Demonstration of Philadelphiachromosome negative abnormal clones in patients with chronic myelogenousleukemia during major cytogenetic responses induced by imatinibmesylate. Leukemia. 2003 March; 17(3):481-7; Hochhaus et al., Molecularand chromosomal mechanisms of resistance to imatinib (STI571) therapy,Leukemia. 2002 November; 16(11):2190-6; O'Dwyer et al., The impact ofclonal evolution on response to imatinib mesylate (STI571) inaccelerated phase CML. Blood. 2002 Sep. 1; 100(5)1628-33; Braziel etal., Hematopathologic and cytogenetic findings in imatinibmesylate-treated chronic myelogenous leukemia patients: 14 months'experience. Blood. 2002 Jul. 15; 100(2):435-41; Corbin et al., Analysisof the structural basis of specificity of inhibition of the Abl kinaseby STI571. J Biol Chem. 2002 Aug. 30; 277(35):32214-9; Wertheim et al.,BCR-ABL-induced adhesion defects are tyrosine kinase-independent. Blood,2002 Jun. 1; 99(11):4122-30; Kantarjian et al., Hematologic andcytogenetic responses to imatinib mesylate in chronic myelogenousleukemia, N Engl J Med. 2002 Feb. 28; 346(9):645-52. Erratum in: N EnglJ Med 2002 Jun. 13; 346(24)1923; Hochhaus et al., Roots of clinicalresistance to STI-571 cancer therapy. Science, 2001 Sep. 21;293(5538):2163; Druker et al., Activity of a specific inhibitor of theBCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemiaand acute lymphoblastic leukemia with the Philadelphia chromosome. NEngl J Med. 2001 Apr. 5; 344(14):1038-42. Erratum in: N Engl J Med 2001Jul. 19; 345(3):232; Mauro et al., Chronic myelogenous leukemia. CurrOpin Oncol. 2001 January; 13(1):3-7. Review; Kolibaba et al., CRKLbinding to BCR-ABL and BCR-ABL transformation. Leuk Lymphoma. 1999March; 33(1-2)119-26; Bhat et al., Interactions of p62(dok) withp210(bcr-abl) and Bcr-Abl-associated proteins. J Biol Chem. 1998 Nov.27; 273(48):32360-8; Senechal et al., Structural requirements forfunction of the Crkl adapter protein in fibroblasts and hematopoieticcells. Mol Cell Biol. 1998 September; 18(9):5082-90; Kolibaba et al.,Protein tyrosine kinases and cancer. Biochim Biophys Acta. 1997 Dec. 9;1333(3):F217-48. Review; Heaney et al., Direct binding of CRKL toBCR-ABL is not required for BCR-ABL transformation. Blood. 1997 Jan. 1;89(1):297-306; Hallek et al., Interaction of the receptor tyrosinekinase p145c-kit with the p210bcr/abl kinase in myeloid cells. Br JHaematol. 1996 July; 94(1):5-16; Oda et al., The SH2 domain of ABL isnot required for factor-independent growth induced by BCR-ABL in amurine myeloid cell line. Leukemia. 1995 February; 9(2):295-301;Carlesso et al., Use of a temperature-sensitive mutant to define thebiological effects of the p210BCR-ABL tyrosine kinase on proliferationof a factor-dependent murine myeloid cell line. Oncogene. 1994 January;9(1):149-56.

Again, we contemplate that compounds of this invention, both asmonotherapies and in combination therapies, will be useful againstleukemias and other cancers including those which are resistant in wholeor part to other anticancer agents, specifically including Gleevec andother kinase inhibitors, and specifically including leukemias involvingone or more mutations in BCR/Abl, within or outside the kinase domain,including but not limited to those noted in any of the foregoingpublications. See in particular Azam et al. and references cited thereinfor examples of such mutations in BCR/Abl, including, among others,mutations in the drug binding cleft, the phosphate binding P loop, theactivation loop, the conserved VAVK of the kinase beta-3 sheet, thecatalytic alpha-1 helix of the small N lobe, the long alpha-3 helixwithin the large C lobe, and the region within the C lobe downstream ofthe activation loop.

Pharmaceutical Methods

The method of the invention comprises administering to a subject in needthereof a therapeutically effective amount of a compound of theinvention.

A “therapeutically effective amount” is that amount effective fordetectable killing or inhibition of the growth or spread of cancercells; the size or number of tumors; or other measure of the level,stage, progression or severity of the cancer. The exact amount requiredwill vary from subject to subject, depending on the species, age, andgeneral condition of the subject, the severity of the disease, theparticular anticancer agent, its mode of administration, combinationtreatment with other therapies, and the like.

The compound, or a composition containing the compound, may beadministered using any amount and any route of administration effectivefor killing or inhibiting the growth of tumors or other forms of cancer.

The anticancer compounds of the invention are preferably formulated indosage unit form for ease of administration and uniformity of dosage.The expression “dosage unit form” as used herein refers to a physicallydiscrete unit of anticancer agent appropriate for the patient to betreated. As is normally the case, the total daily usage of the compoundsand compositions of the present invention will be decided by theattending a physician using routine reliance upon sound medicaljudgment. The specific therapeutically effective dose level for anyparticular patient or organism will depend upon a variety of factorsincluding the disorder being treated; the severity of the disorder; thepotency of the specific compound employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the route and schedule of administration; the rate ofmetabolism and/or excretion of the compound; the duration of thetreatment; drugs used in combination or coincident with administrationof the compound of this invention; and like factors well known in themedical arts.

Furthermore, after formulation with an appropriate pharmaceuticallyacceptable carrier in a desired dosage, the compositions of thisinvention can be administered to humans and other animals orally,rectally, parenterally, intracisternally, intravaginally,intraperitoneally, topically (as by transdermal patch, powders,ointments, or drops), sublingually, bucally, as an oral or nasal spray,or the like.

The effective systemic dose of the compound will typically be in therange of 0.01 to 500 mg of compound per kg of patient body weight,preferably 0.1 to 125 mg/kg, and in some cases 1 to 25 mg/kg,administered in single or multiple doses. Generally, the compound may beadministered to patients in need of such treatment in a daily dose rangeof about 50 to about 2000 mg per patient. Administration may be once ormultiple times daily, weekly (or at some other multiple-day interval) oron an intermittent schedule. For example, the compound may beadministered one or more times per day on a weekly basis (e.g. everyMonday) indefinitely or for a period of weeks, e.g. 4-10 weeks.Alternatively, it may be administered daily for a period of days (e.g.2-10 days) followed by a period of days (e.g. 1-30 days) withoutadministration of the compound, with that cycle repeated indefinitely orfor a given number of repititions, e.g. 4-10 cycles. As an example, acompound of the invention may be administered daily for 5 days, thendiscontinued for 9 days, then administered daily for another 5 dayperiod, then discontinued for 9 days, and so on, repeating the cycleindefinitely, or for a total of 4-10 times.

The amount of compound which will be effective in the treatment orprevention of a particular disorder or condition will depend in part onwell known factors affecting drug dosage. In addition, in vitro or inviva assays may optionally be employed to help identify optimal dosageranges. A rough guide to effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.The precise dosage level should be determined by the attending physicianor other health care provider and will depend upon well known factors,including route of administration, and the age, body weight, sex andgeneral health of the individual; the nature, severity and clinicalstage of the disease; the use (or not) of concomitant therapies; and thenature and extent of genetic engineering of cells in the patient.

When administered for the treatment or inhibition of a particulardisease state or disorder, the effective dosage of the compound of thisinvention may vary depending upon the particular compound utilized, themode of administration, the condition, and severity thereof, of thecondition being treated, as well as the various physical factors relatedto the individual being treated. In many cases, satisfactory results maybe obtained when the compound is administered in a daily dosage of fromabout 0.01 mg/kg-500 mg/kg, preferably between 0.1 and 125 mg/kg, andmore preferably between 1 and 25 mg/kg. The projected daily dosages areexpected to vary with route of administration. Thus, parenteral dosingwill often be at levels of roughly 10% to 20% of oral dosing levels.

When the compound of this invention is used as part of a combinationregimen, dosages of each of the components of the combination areadministered during a desired treatment period. The components of thecombination may administered at the same time; either as a unitarydosage form containing both components, or as separate dosage units; thecomponents of the combination can also be administered at differenttimes during a treatment period, or one may be administered as apretreatment for the other.

Regarding the Compounds

Compounds of present invention can exist in free form for treatment, orwhere appropriate, as a pharmaceutically acceptable salt or otherderivative. As used herein, the term “pharmaceutically acceptable salt”refers to those salts which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of humans andlower animals without undue toxicity, irritation, allergic response andthe like, and are commensurate with a reasonable benefit/risk ratio,Pharmaceutically acceptable salts of amines, carboxylic acids,phosphonates and other types of compounds, are well known in the art.For example, S. M. Berge, at al. describe pharmaceutically acceptablesalts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977),incorporated herein by reference. The salts can be prepared in situduring the isolation and purification of the compounds of the invention,or separately by reacting the free base or free acid of a compound ofthe invention with a suitable base or acid, respectively. Examples ofpharmaceutically acceptable, nontoxic acid addition salts are salts ofan amino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, oxalic acid, maleic acid,tartaric acid, citric acid, succinic acid or malonic acid or by usingother methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hernisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like.Further pharmaceutically acceptable salts include, when appropriate,nontoxic ammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, loweralkyl sulfonate and aryl sulfonate.

Additionally, as used herein, the term “pharmaceutically acceptableester” refers preferably to esters which hydrolyze in vivo and includethose that break down readily in the human body to leave the parentcompound or a salt thereof. Suitable ester groups include, for example,those derived from pharmaceutically acceptable aliphatic carboxylicacids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioicacids, in which each alkyl or alkenyl moiety advantageously has not morethan 6 carbon atoms. Examples of particular esters include formates,acetates, propionates, butyrates, acrylates and ethylsuccinates.Obviously, esters can be formed with a hydroxyl or carboxylic acid groupof the compound of the invention.

Furthermore, the term “pharmaceutically acceptable prodrugs” as usedherein refers to those prodrugs of the compounds of the presentinvention which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswith undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use, as well as the zwitterionic forms, where possible,of the compounds of the invention. The term “prodrug” refers tocompounds that are transformed in vivo to yield the parent compound ofthe above formula, for example by hydrolysis in blood. See, e.g., T.Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 ofthe A.C.S. Symposium Series, and Edward B. Roche, ed., BioreversibleCarriers in Drug Design, American Pharmaceutical Assocn. and PergamonPress, 1987, both of which are incorporated herein by reference.

Compositions

Compositions are provided, which comprise any one of the compoundsdescribed herein (or a prodrug, pharmaceutically acceptable salt orother pharmaceutically acceptable derivative thereof), and one or morepharmaceutically acceptable carriers or excipients. These compositionsoptionally further comprise one or more additional therapeutic agents.Alternatively, a compound of this invention may be administered to apatient in need thereof in combination with the administration of one ormore other therapeutic regimens (e.g. Gleevec or other kinaseinhibitors, interferon, bone marrow transplant, farnesyl transferaseinhibitors, bisphosphonates, thalidomide, cancer vaccines, hormonaltherapy, antibodies, radiation, etc). For example, additionaltherapeutic agents for conjoint administration or inclusion in apharmaceutical composition with a compound of this invention may beanother one or more anticancer agents.

As described herein, the compositions of the present invention comprisea compound of the invention together with a pharmaceutically acceptablecarrier, which, as used herein, includes any and all solvents, diluents,or other vehicle, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, solidbinders, lubricants and the like, as suited to the particular dosageform desired. Remington's Pharmaceutical Sciences, Fifteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1975) discloses variouscarriers used in formulating pharmaceutical compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutical composition, it's use is contemplatedto be within the scope of this invention. Some examples of materialswhich can serve as pharmaceutically acceptable carriers include, but arenot limited to, sugars such as lactose, glucose and sucrose; starchessuch as corn starch and potato starch; cellulose and its derivativessuch as sodium carboxymethyl cellulose, ethyl cellulose and celluloseacetate; powdered tragacanth; malt; gelatin; talc; excipients such ascocoa butter and suppository waxes; oils such as peanut oil, cottonseedoil; safflower oil; sesame oil; olive oil; corn oil and soybean oil;glycols; such a propylene glycol; esters such as ethyl oleate and ethyllaurate; agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition.

Formulations

This invention also encompasses a class of compositions comprising theactive compounds of this invention in association with one or morepharmaceutically-acceptable carriers and/or diluents and/or adjuvants(collectively referred to herein as “carrier” materials) and, ifdesired, other active ingredients. The active compounds of the presentinvention may be administered by any suitable route, preferably in theform of a pharmaceutical composition adapted to such a route, and in adose effective for the treatment intended. The compounds andcompositions of the present invention may, for example, be administeredorally, mucosally, topically, rectally, pulmonarily such as byinhalation spray, or parentally including intravascularly,intravenously, intraperitoneally, subcutaneously, intramuscularly,intrasternally and infusion techniques, in dosage unit formulationscontaining conventional pharmaceutically acceptable carriers, adjuvants,and vehicles.

The pharmaceutically active compounds of this invention can be processedin accordance with conventional methods of pharmacy to produce medicinalagents for administration to patients, including humans and othermammals.

For oral administration, the pharmaceutical composition may be in theform of, for example, a tablet, capsule, suspension or liquid. Thepharmaceutical composition is preferably made in the form of a dosageunit containing a particular amount of the active ingredient.

Examples of such dosage units are tablets or capsules. For example,these may contain an amount of active ingredient from about 1 to 2000mg, preferably from about 1 to 500 mg, more commonly from about 5 to 200mg. A suitable daily dose for a human or other mammal may vary dependingon the condition of the patient and other factors, but, once again, canbe determined using routine methods.

The amount of compounds which are administered and the dosage regimenfor treating a disease condition with the compounds and/or compositionsof this invention depends on a variety of factors, including the age,weight, sex and medical condition of the subject, the type of disease,the severity of the disease, the route and frequency of administration,and the particular compound employed. Thus, the dosage regimen may varywidely, but can be determined routinely using standard methods. Atypical daily dose is in the range of 0.01 to 500 mg of compound per kgbody weight, preferably between 0.1 and 125 mg/kg body weight and insome cases between 1 and 25 mg/kg body weight. As mentioned previously,the daily dose can be given in one administration or may be dividedbetween 2, 3, 4 or more administrations.

For therapeutic purposes, the active compounds of this invention areordinarily combined with one or more adjuvants, excipients or carriersappropriate to the indicated route of administration. If administeredper os, the compounds may be admixed with lactose, sucrose, starchpowder, cellulose esters of alkanoic acids, cellulose alkyl esters,talc, stearic acid, magnesium stearate, magnesium oxide, sodium andcalcium salts of phosphoric and sulfuric acids, gelatin, acacia gum,sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, andthen tableted or encapsulated for convenient administration. Suchcapsules or tablets may contain a controlled-release formulation as maybe provided in a dispersion of active compound in hydroxypropy1methylcellulose.

In the case of skin conditions, it may be preferable to apply a topicalpreparation of compounds of this invention to the affected area two tofour times a day.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin(e.g., liniments, lotions, ointments, creams, or pastes) and dropssuitable for administration to the eye, ear, or nose. A suitable topicaldose of active ingredient of a compound of _(t)he invention is 0.1 mg to150 mg administered one to four, preferably one or two times daily. Fortopical administration, the active ingredient may comprise from 0.001%to 10% w/w, e.g., from 1% to 2% by weight of the formulation, althoughit may comprise as much as 10% w/w, but preferably not more than 5% w/w,and more preferably from 0.1% to 1% of the formulation.

When formulated in an ointment, the active ingredients may be employedwith either paraffinic or a water-miscible ointment base. Alternatively,the active ingredients may be formulated in a cream with an oil-in-watercream base. if desired, the aqueous phase of the cream base may include,for example at Least 30% w/w of a polyhydric alcohol such as propyleneglycol, butane-1,3-diol, mannitol, sorbitol, glycerol, polyethyleneglycol and mixtures thereof. The topical formulation may desirablyinclude a compound which enhances absorption or penetration of theactive ingredient through the skin or other affected areas. Examples ofsuch dermal penetration enhancers include dimethylsulfoxide and relatedanalogs.

The compounds of this invention can also be administered by atransdermal device. Preferably transdermal administration will beaccomplished using a patch either of the reservoir and porous membranetype or of a solid matrix variety. In either case, the active agent isdelivered—continuously from the reservoir or microcapsules through amembrane into the active agent permeable adhesive, which is in contactwith the skin or mucosa of the recipient. If the active agent isabsorbed through the skin, a controlled and predetermined flow of theactive agent is administered to the recipient. In the case ofmicrocapsules, the encapsulating agent may also function as themembrane.

The oily phase of the emulsions of this invention may be constitutedfrom known ingredients in a known manner. While the phase may comprisemerely an emulsifier, it may comprise a mixture of at least oneemulsifier with a fat or an oil or with both a fat and an oil.Preferably, a hydrophilic emulsifier is included together with alipophilic emulsifier which acts as a stabilizer. It is also preferredto include both an oil and a fat. Together, the emulsifier(s) with orwithout stabilizer(s) make-up the socalled emulsifying wax, and the waxtogether with the oil and fat make up the so-called emulsifying ointmentbase which forms the oily dispersed phase of the cream formulations.Emulsifiers and emulsion stabilizers suitable for use in the formulationof the present invention include Tween 60, Span 80, cetostearyl alcohol,myristyl alcohol, glyceryl monostearate, sodium lauryl sulfate, glyceryldistearate alone or with a wax, or other materials well known in theart.

The choice of suitable oils or fats for the formulation is based onachieving the desired cosmetic properties, since the solubility of theactive compound in most oils likely to be used in pharmaceuticalemulsion formulations is very low. Thus, the cream should preferably bea non-greasy, non-staining and washable product with suitableconsistency to avoid leakage from tubes or other containers. Straight orbranched chain, mono- or dibasic alkyl esters such as di-isoadipate,isocetyl stearate, propylene glycol diester of coconut fatty acids,isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate,2-ethylhexyl palmitate or a blend of branched chain esters may be used.These may be used alone or in combination depending on the propertiesrequired.

Alternatively, high melting point lipids such as white soft paraffinand/or liquid paraffin or other mineral oils can be used.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredients are dissolved or suspended insuitable carrier, especially an aqueous solvent for the activeingredients. The active ingredients are preferably present in suchformulations in a concentration of 0.5 to 20%, advantageously 0.5 to 10%and particularly about 1.5% w/w.

Formulations for parenteral administration may be in the form of aqueousor non-aqueous isotonic sterile injection solutions or suspensions.These solutions and suspensions may be prepared from sterile powders orgranules using one or more of the carriers or diluents mentioned for usein the formulations for oral administration or by using other suitabledispersing or wetting agents and suspending agents. The compounds may bedissolved in water, polyethylene glycol, propylene glycol, ethanol, cornoil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodiumchloride, tragacanth gum, and/or various buffers.

Other adjuvants and modes of administration are well and widely known inthe pharmaceutical art. The active ingredient may also be administeredby injection as a composition with suitable carriers including saline,dextrose, or water, or with cyclodextrin (i.e. Captisol), cosolventsolubilization (i.e. propylene glycol) or micellar solubilization (i.e.Tween 80).

The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution, and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employed,including synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

For pulmonary administration, the pharmaceutical composition may beadministered in the form of an aerosol or with an inhaler including drypowder aerosol.

Suppositories for rectal administration of the drug can be prepared bymixing the drug with a suitable nonirritating excipient such as cocoabutter and polyethylene glycols that are solid at ordinary temperaturesbut liquid at the rectal temperature and will therefore melt in therectum and release the drug.

The pharmaceutical compositions may be subjected to conventionalpharmaceutical operations such as sterilization and/or may containconventional adjuvants, such as preservatives, stabilizers, wettingagents, emulsifiers, buffers etc. Tablets and pills can additionally beprepared with enteric coatings. Such compositions may also compriseadjuvants, such as wetting, sweetening, flavoring, and perfuming agents.

Pharmaceutical compositions of this invention comprise a compound of theformulas described herein or a pharmaceutically acceptable salt thereof;an additional agent selected from a kinase inhibitory agent (smallmolecule, polypeptide, antibody, etc.), an immunosuppressant, ananticancer agent, an anti-viral agent, antiinflammatory agent,antifungal agent, antibiotic, or an anti-vascular hyperproliferationcompound; and any pharmaceutically acceptable carrier, adjuvant orvehicle.

Alternate compositions of this invention comprise a compound of theformulae described herein or a pharmaceutically acceptable salt thereof;and a pharmaceutically acceptable carrier, adjuvant or vehicle. Suchcompositions may optionally comprise one or more additional therapeuticagents, including, for example, kinase inhibitory agents (smallmolecule, polypeptide, antibody, etc.), immunosuppressants, anti-canceragents, anti-viral agents, antiinflammatory agents, antifungal agents,antibiotics, or anti-vascular hyperproliferation compounds.

The term “pharmaceutically acceptable carrier or adjuvant” refers to acarrier or adjuvant that may be administered to a patient, together witha compound of this invention, and which does not destroy thepharmacological activity thereof and is nontoxic when administered indoses sufficient to deliver a therapeutic amount of the compound.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions of this invention include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, selfemulsifying drug delivery systems (SEDDS) such asd-atocopherol polyethyleneglycol 1000 succinate, surfactants used inpharmaceutical dosage forms such as Tweens or other similar polymericdelivery matrices, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbets,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Cyclodextrins such as u-, P-, and y-cyclodextrin, orchemically modified derivatives such as hydroxyalkylcyclodextrins,including 2 and 3-hydroxypropyl-cyclodextrins, or other solubilizedderivatives may also be advantageously used to enhance delivery ofcompounds of the formulae described herein.

The pharmaceutical compositions may be orally administered in any orallyacceptable dosage form including, but not limited to, capsules, tablets,emulsions and aqueous suspensions, dispersions and solutions. In thecase of tablets for oral use, carriers which are commonly used includelactose and corn starch. Lubricating agents, such as magnesium stearate,are also typically added. For oral administration in a capsule form,useful diluents include lactose and dried corn starch. When aqueoussuspensions and/or emulsions are administered orally, the activeingredient may be suspended or dissolved in an oily phase is combinedwith emulsifying and/or suspending agents.

If desired, certain sweetening, flavoring and/or coloring agents may beadded. The pharmaceutical compositions may comprise formulationsutilizing liposome or microencapsulation techniques, various examples ofwhich are known in the art.

The pharmaceutical compositions may be administered by nasal aerosol orinhalation. Such compositions are prepared according to techniques wellknown in the art of pharmaceutical formulation and may be prepared assolutions in saline, employing benzyl alcohol or other suitablepreservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents, examplesof which are also well known in the art.

Combinations

While the compounds of the invention can be administered as the soleactive pharmaceutical agent, they can also be used in combination withone or more other compounds of the invention or with one or more otheragents. When administered as a combination, the therapeutic agents canbe formulated as separate compositions that are administered at the sametime or sequentially at different times, or the therapeutic agents canbe given as a single composition.

The phrase “combination therapy”, in referring to the use of a compoundof this invention together with another pharmaceutical agent, means thecoadministration of each agent in a substantially simultaneous manner aswell as the administration of each agent in a sequential manner, ineither case, in a regimen that will provide beneficial effects of thedrug combination. Coadministration includes inter alia the simultaneousdelivery, e.g., in a single tablet, capsule, injection or other dosageform having a fixed ratio of these active agents, as well as thesimultaneous delivery in multiple, separate dosage forms for each agentrespectively.

Thus, the administration of compounds of the present invention may be inconjunction with additional therapies known to those skilled in the artin the prevention or treatment of cancer, such as radiation therapy orcytostatic agents, cytotoxic agents, other anti-cancer agents and otherdrugs to amerliorate symptoms of the cancer or side effects of any ofthe drugs.

If formulated as a fixed dose, such combination products employ thecompounds of this invention within the accepted dosage ranges. Compoundsof this invention may also be administered sequentially with otheranticancer or cytotoxic agents when a combination formulation isinappropriate. The invention is not limited in the sequence ofadministration; compounds of this invention may be administered priorto, simulateously with, or after administration of the other anticanceror cytotoxic agent.

Currently, standard treatment of primary tumors consists of surgicalexcision, when appropriate, followed by either radiation orchemotherapy, typically administered intravenously (IV). The typicalchemotherapy regime consists of either DNA alkylating agents, DNAintercalating agents, CDK inhibitors, or microtubule poisons. Thechemotherapy doses used are just below the maximal tolerated dose andtherefore dose limiting toxicities typically include, nausea, vomiting,diarrhea, hair loss, neutropenia and the like.

There are large numbers of antineoplastic agents available in commercialuse, in clinical evaluation and in pre-clinical development, which wouldbe selected for treatment of cancer by combination drug chemotherapy.And there are several major categories of such antineoplastic agents,namely, antibiotic-type agents, alkylating agents, antimetaboliteagents, hormonal agents, immunological agents, interferon-type agentsand a category of miscellaneous agents.

A first family of antineoplastic agents which may be used in combinationwith compounds of the present invention includesantimetabolite-type/thymidilate synthase inhibitor antineoplasticagents. Suitable antimetabolite antineoplastic agents may be selectedfrom but not limited to the group consisting of 5-FU-fibrinogen,acanthifolic acid, aminothiadiazole, brequinar sodium, carmofur,CibaGeigy CGP-30694, cyclopentyl cytosine, cytarabine phosphatestearate, cytarabine conjugates, Lilly DATHF, Merrel Dow DDFC,dezaguanine, dideoxycytidine, dideoxyguanosine, didox, Yoshitomi DMDC,doxifluridine, Wellcome EHNA, Merck & Co.

EX-015, fazarabine, floxuridine, fludarabine phosphate, 5fluorouracil,N-(21-furanidyl) fluorouracil, Dalichi Seiyaku FO-152, isopropylpyrrolizine, Lilly LY-188011, Lilly LY-264618, methobenzaprim,methotrexate, Wellcome MZPES, norspermidine, NCI NSC-127716, NCINSC-264880, NCI NSC-39661, NCI NSC-612567, Warner-Lambert PALA,pentostatin, piritrexim, plicamycin, Asahi Chemical PL-AC, TakedaTAC788, thioguanine, tiazofurin, Erbamont TIF, trimetrexate, tyrosinekinase inhibitors, Taiho UFT and uricytin.

A second family of antineoplastic agents which may be used incombination with compounds of the present invention consists ofalkylating-type antineoplastic agents. Suitable alkylating-typeantineoplastic agents may be selected from but not limited to the groupconsisting of Shlonogi 254-S, aldo-phosphamide analogues, altretamine,anaxirone, Boehringer Mannheim BBR-2207, bestrabucil, budotitane,Wakunaga CA-102, carboplatin, carmustine. Chinoin-139, Chinoin-153,chlorambucil, cisplatin, cyclophosphamide, American Cyanamid CL-286558,Sanofi CY-233, cyplatate, Degussa D 384, Sumimoto DACHP(Myr)2,diphenylspiromustine, diplatinum cytostatic, Erba distamycinderivatives, Chugai DWA-2114R, ITI E09, elmustine, Erbamont FCE-24517,estramustine phosphate sodium, fotemustine, Unimed G M, ChinoinGYKI-17230, hepsulfam, ifosfamide, iproplatin, lomustine, mafosfamide,mitolactolf Nippon Kayaku NK-121, NCI NSC-264395, NCI NSC-342215,oxaliplatin, Upjohn PCNU, prednimustine, Proter PTT-119, ranimustine,semustine, SmithKline SK&F-101772, Yakult Honsha SN-22, spiromus-tine,Tanabe Seiyaku TA-077, tauromustine, temozolomide, teroxirone,tetraplatin and trimelamol.

A third family of antineoplastic agents which may be used in combinationwith compounds of the present invention consists of antibiotic-typeantineoplastic agents. Suitable antibiotic-type antineoplastic agentsmay be selected from but not limited to the group consisting of Taiho4181-A, aclarubicin, actinomycin D, actinoplanone, Erbamont ADR-456,aeroplysinin derivative, Ajinomoto AN II, Ajinomoto AN3, Nippon Sodaanisomycins, anthracycline, azino-mycin-A, bisucaberin, Bristol-MyersBL-6859, Bristol-Myers BMY-25067, Bristol-Myers BNY-25551, Bristol-MyersBNY-26605 IBristolMyers BNY-27557, Bristol-Myers BMY-28438, bleomycinsulfate, bryostatin-1, Taiho C-1027, calichemycin, chromoxlmycin,dactinomycin, daunorubicin, Kyowa Hakko DC-102, Kyowa Hakko DC-79, KyowaHakko DC-88A, Kyowa Hakko, 0089-Al, Kyowa Hakko DC92-B, ditrisarubicinB, Shionogi DOB-41, doxorubicin, doxorubicin-fibrinogen, elsamicin-A,epirubicin, erbstatin, esorubicin, esperamicin-Al, esperamicin-Alb,Erbamont FCE21954, Fujisawa FK-973, fostriecin, Fujisawa FR-900482,glidobactin, gregatin-A, grincamycin, herbimycin, idarubicin, illudins,kazusamycin, kesarirhodins, Kyowa Hakko KM-5539, Kirin Brewery KRN-8602,Kyowa Hakko KT-5432, Kyowa Hakko KT-5694, Kyowa Hakko KT-6149, AmericanCyanamid LL-D49194, Meiji Seika ME 2303, menogaril, mitomycin,mitoxantrone, SmithKline M-TAG, neoenactin, Nippon Kayaku NK-313, NipponKayaku NKT-01, SRI International NSC-357704, oxalysine, oxaunomycin,peplomycin, pilatin, pirarubicin, porothramycin, pyrindanycin A, TobishiRA-I, rapamycin, rhizoxin, rodorubicin, sibanomicin, siwenmycin,Sumitomo SM5887, Snow Brand SN-706, Snow Brand SN-07, sorangicin-A,sparsomycin, SS Pharmaceutical SS-21020, SS Pharmaceutical SS-7313B, SSPharmaceutical SS-9816B, steffimycin B, Taiho 4181-2, talisomycin,Takeda TAN-868A, terpentecin, thrazine, tricrozarin A, Upjohn U-73975,Kyowa Hakko UCN-10028A, Fujisawa WF-3405, Yoshitomi Y-25024 andzorubicin.

A fourth family of antineoplastic agents which may be used incombination with compounds of the present invention consists of amiscellaneous family of antineoplastic agents, including tubulininteracting agents, topoisomerase II inhibitors, topoisomerase Iinhibitors and hormonal agents, selected from but not limited to thegroup consisting of (xcarotene, (X-difluoromethyl-arginine, acitretin,Biotec AD-5, Kyorin AHC-52, alstonine, amonafide, amphethinile,amsacrine, Angiostat, ankinomycin, anti-neoplaston A10, antineoplastonA2, antineoplaston A3, antineoplaston A5. antineoplaston AS2-1F HenkelAPD, aphidicolin glycinate, asparaginase, Avarol, baccharin, batracylin,benfluron, benzotript, Ipsen-Beaufour BIM-23015, bisantrene, BristoMyersBNY-40481, Vestar boron-10, bromofosfamide, Wellcome BW-502, WellcomeBW-773, caracemide, carmethizole hydrochloride, Ajinomoto CDAF,chlorsulfaquinoxalone, Chemes CHX-2053, Chemex CHX-100, Warner-LambertCI-921, WarnerLambert CI-937, Warner-Lambert CI-941, Warner-LambertCI958, clanfenur, claviridenone, ICN compound 1259, ICN compound 4711,Contracan, Yakult Honsha CPT-11, crisnatol, curaderm, cytochalasin B.cytarabine, cytocytin, Merz D-609, DAMS maleate, dacarbazine,datelliptinium, didemnin-B, dihaematoporphyrin ether, dihydrolenperone,dinaline, distamycin, Toyo Pharmar DM-341, Toyo Pharmar DM-75, DailchiSeiyaku DN-9693, docetaxel elliprabin, elliptinium acetate, TsumuraEPMTC, the epothilones, ergotamine, etoposide, etretinate, fenretinide,Fujisawa FR-57704t gallium nitrate, genkwadaphnin, Chugai GLA-43, GlaxoGR-63178, grifolan NMF5N, hexadecylphosphocholine, Green Cross HO-221,homoharringtonine, hydroxyurea, BTG ICRF-187, ilmofosine, isoglutamine,isotretinoin, Otsuka JI-36, Ramat K-477, Otsuak K-76COONa, KurehaChemical K-AM, MECT Corp KI-8110, American Cyanamid L-623, leukoregulin,lonidamine, Lundbeck LU 1121 Lilly LY-186641, NCI (US) MAP, marycin,Merrel Dow MDL-27048, Medco MEDR-340, merbarone, merocyaninederivatives, methylanilinoacridine, Molecular Genetics MGI136,minactivin, mitonafide, mitoquidone mopidamol, motretinide, ZenyakuKogyo MST-16, N-(retinoyl)amino acids, Nisshin Flour Milling N-021,N-acylated-dehydroalanines, nafazatrom, Taisho NCU-190, nocodazolederivative, Normosang, NCI NSC-145813, NCI NSC-361456, NCI NSC-604782,NCI NSC-95580, ocreotide, Ono ONO-112, oquizanocine, Akzo Org-10172,paclitaxel, pancratistatin, pazelliptine, WarnerLambert PD-111707,Warner-Lambert PD-115934, Warner-Lambert PD-131141, Pierre FabrePE-1001, ICRT peptide D, piroxantrone, polyhaematoporphyrin, polypreicacid, Eternal porphyrin, probimane, procarbazine, proglumide, invitronprotease nexin I, Tobishi RA-700, razoxane, Sapporo Breweries RBS,restrictin-P, retelliptine, retinoic acid, Rhone-Poulenc RP-49532,Rhone-Poulenc RP-56976, SmithKline SK&F-104864, Sumitomo SM-108, KuraraySMANCS, SeaPharm SP10094, spatol, spirocyclopropane derivatives,spirogermanium, Unimed, SS Pharmaceutical SS-554, strypoldinone,Stypoldione, Suntory SUN 0237, Suntory SUN 2071, superoxide dismutase,Toyama T-506, Toyama T-680, taxol, Teijin TEI-0303, teniposide,thaliblastine, Eastman Kodak TJB-29, tocotrienol, topotecan, Topostin,Teijin TT82, Kyowa Hakko UCN-01, Kyowa Hakko UCN-1028, ukrain, EastmanKodak USB-006, vinblastine sulfate, vincristine, vindesine,vinestramide, vinorelbine, vintriptol, vinzolidine, withanolides andYamanouchi YM Alternatively, the present compounds may also be used inco-therapies with other anti-neoplastic agents, such as acemannan,aclarubicin, aldesleukin, alemtuzumab, alitretinoin, altretamine,amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide,anastrozole, ANCER, ancestim, ARGLABIN, arsenic trioxide, BAM 002(Novelos), bexarotene, bicalutamide, broxuridine, capecitabine,celmoleukin, cetrorelix, cladribine, clotrimazole, cytarabine ocfosfate,DA 3030 (Dong-A), daclizumab, denileukin diftitox, desiorelin,dexrazoxane, dilazep, docetaxel, docosanol, doxercalciferol,doxifluridine, doxorubicin, bromocriptine, carmustine, cytarabine,fluorouracil, HIT diclofenac, interferon alfa, daunorubicin,doxorubicin, tretinoin, edelfosine, edrecolomab eflornithine, emitefur,epirubicin, epoetin beta, etoposide phosphate, exemestane, exisulind,fadrozole, filgrastim, finasteride, fludarabine phosphate, formestane,fotemustine, gallium nitrate, gemcitabine, gemtuzumab zogamicin,gimeracil/oteracil/tegafur combination, glycopine, goserelin,heptaplatin, human chorionic gonadotropin, human fetal alphafetoprotein, ibandronic acid, idarublcin, (imiquimod, interferon alfa,interferon alfa, natural, interferon alfa-2, interferon alfa-2a,interferon alfa-2b, interferon alfa-NI, interferon alfa-n3, interferonalfacon1, interferon alpha, natural, interferon beta, interferonbeta-Ia, interferon beta-Ib, interferon gamma, natural interferongamma-Ia, interferon gamma-Ib, interleukin-I beta, iobenguane,irinotecan, irsogladine, lanreotide, LC 9018 (Yakult), leflunomide,lenograstim, lentinan sulfate, letrozole, leukocyte alpha interferon,leuprorelin, levamisole+fluorouracil, liarozole, lobaplatin, lonidamine,lovastatin, masoprocol, melarsoprol, metoclopramide, mifepristone,miltefosine, mirimostim, mismatched double stranded RNA, mitoguazone,mitolactol, mitoxantrone, molgramostim, nafarelin, naloxone+pentazocine,nartograstim, nedaplatin, nilutamide, noscapine, novel erythropoiesisstimulating protein, NSC 631570 octreotide, oprelvekin, osaterone,oxaliplatin, paclitaxel, pamidronic acid, pegaspargase, peginterferonalfa-2b, pentosan polysulfate sodium, pentostatin, picibanil,pirarubicin, rabbit antithymocyte polyclonal antibody, polyethyleneglycol interferon alfa-2a, porfimer sodium, raloxifene, raltitrexed,rasburlcase, rhenium Re 186 etidronate, RII retinamide, rituximab,romurtide, samarium (153 Sm) lexidronam, sargramostim, sizofiran,sobuzoxane, sonermin, strontium-89 chloride, suramin, tasonermin,tazarotene, tegafur, temoporfin, temozolomide, teniposide,tetrachlorodecaoxide, thalidomide, thymalfasin, thyrotropin alfa,topotecan, toremifene, tositumomab-iodine 131, trastuzumab, treosulfan,tretinoin, trilostane, trimetrexate, triptorelin, tumor necrosis factoralpha, natural, ubenimex, bladder cancer vaccine, Maruyama, vaccine,melanoma lysate vaccine, valrubicin, verteporfin, vinorelbine,VIRULIZIN, zinostatin stimalamer, or zoledronic acid; abarelix; AE 941(Aetema), ambamustine, antisense oligonucleotide, bcl-2 (Genta), APC8015 (Dendreon), cetuximab, decitabine, dexaminoglutethimide,diaziquone, EL 532 (Elan), EM 800 (Endorecherche), eniluracil,etanidazole, fenretinidel filgrastim SDO1 (Amgen), fulvestrant,galocitabine, gastrin 17 immunogen, HLA-B7 gene therapy (Vical),granulocyte macrophage colony stimulating factor, histaminedihydrochloride, ibritumomab tiuxetan, ilomastat, IM 862 (Cytran),interleukin iproxifene, LDI 200 (Milkhaus), leridistim, lintuzumab, CA125 MAb (Biomira), cancer MAb (Japan Pharmaceutical Development), HER-2and Fe MAb (Medarex), idiotypic 105AD7 MAb (CRC Technology), idiotypicCEA MAb (Trilex), LYM iodine 131 MAb (Techniclone), polymorphicepithelial mucin-yttrium 90 MAb (Antisoma), marimastat, menogaril,mitumomab, motexafin, gadolinium, MX 6 (Galderma), nelarabine,nolatrexed, P 30 protein, pegvisomant, pemetrexed, porfiromycin,prinomastat, RL 0903 (Shire), rubitecan, satraplatin, sodiumphenylacetate, sparfosic acid, SRL 172 (SR Pharma), SU 5418 (SUGEN)y SU6668 (SUGEN), TA 077 (Tanabe), tetrathiomolybdate, thaliblastine,thrombopoietin, tin ethyl etiopurpurin, tirapazamine, cancer vaccine(Biomira), melanoma vaccine (New York University), melanoma vaccine(Sloan Kettering Institute), melanoma oncolysate vaccine (New YorkMedical College), viral melanoma cell lysates vaccine (Royal NewcastleHospital), or valspodar.

Treatment Kits

In other embodiments, the present invention relates to a kit forconveniently and effectively carrying out the methods in accordance withthe present invention. In general, the pharmaceutical pack or kitcomprises one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention. Suchkits are especially suited for the delivery of solid oral forms such astablets or capsules. Such a kit preferably includes a number of unitdosages, and may also include a card having the dosages oriented in theorder of their intended use. If desired, a memory aid can be provided,for example in the form of numbers, letters, or other markings or with acalendar insert, designating the days in the treatment schedule in whichthe dosages can be administered. Optionally associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticalproducts, which notice reflects approval by the agency of manufacture,use or sale for human administration.

The following representative examples contain important additionalinformation, exemplification and guidance which can be adapted to thepractice of this invention in its various embodiments and theequivalents thereof. These examples are intended to help illustrate theinvention, and are not intended to, nor should they be construed to,limit its scope. Indeed, various modifications of the invention, andmany further embodiments thereof, in addition to those shown anddescribed herein, will become apparent to those skilled in the art uponreview of this document, including the examples which follow and thereferences to the scientific and patent literature cited herein. Thecontents of those cited references are incorporated herein by referenceto help illustrate the state of the art. In addition, for purposes ofthis invention, the chemical elements are identified in accordance withthe Periodic Table of the Elements, CAS version, Handbook of Chemistryand Physics, 75^(th) Ed., inside cover. Additionally, general principlesof organic chemistry, as well as specific functional moieties andreactivity, are described in “Organic Chemistry”, Thomas Sorrell,University Science Books, Sausalito: 1999, and “Organic Chemistry”,Morrison & Boyd (3d Ed), the entire contents of both of which areincorporated herein by reference.

EXAMPLES

Some of the following compounds have been converted into HCl salt. Thegeneral procedure for generating HCl salts is described below:

To the final product was added Just enough MeOH saturated with HCl (g)to dissolve, cooled to 0° C. for 0.5-1 h, filtered, washed solid withice cold MeOH then Et₂O, and the resulting solid dried in a vacuumdesiccator to provide in most cases the tris HCl salt.

Example 11-(1H-Pyrazole-1-carboxamide-N-methyl)-4-methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)benzamide

1-(Bromomethyl)-4-nitro-2-(trifluoromethyl)benzene: A suspension of2-methyl-5-nitrobenzotrifluoride (3.90 g, 19 mmol), N-bromosuccinimide(NBS, 3.56 g, 20 mmol), 2,2′-azobis(2-methylpropionitrile) (AIBN, 94 mg,0.6 mmol) in CCl₄ (40 mL) was refluxed under N₂ for 16 h. HPLC indicatedca. 50% conversion. More NBS (10 mmol) and AIBN (0.6 mmol) was added,and the mixture was refluxed for another 14 h. HPLC indicated ca. 80%conversion. The reaction mixture was cooled down, and the solid wasfiltered off and washed with EtOAc. The combined filtrate was washedwith aq. NaHCO₃, dried over Na₂SO₄, filtered, concentrated on rotovapand further dried under vacuum. 1H NMR shows the ratio of desiredproduct to unreacted 2-methyl-5-nitrobenzotrifluoride is 75:25. Thismaterial was not purified but used directly in the next step.

1-Methyl-4-(4-nitro-2-(trifluoromethyl)benzyl)piperazine: To a solutionof crude 1-(bromomethyl)-4-nitro-2-(trifluoromethyl)benzene (13.33 mmol,75% pure) in DCM (10 mL) was added Et₃N (1.4 mL, 10 mmol) and1-methylpiperazine (1.1 mL, 10 mmol). After stirring for 3 h at rt, aq.NaHCO₃ was added, and the mixture was extracted with DCM. The combinedorganic layer was dried over Na₂SO₄, filtered, concentrated, and theresulting residue was purified by silica gel chromatography (eluted with10% MeOH/DCM) to provide 2.21 g of product as a pale yellow oil.

4((4-Methylpiperazin-1-yl)methyl-3-(trifluoromethyl)aniline: Asuspension of 1-methyl-4-(4-nitro-2-(trifluoromethyl)benzyl)piperazine(1.23 g, 4 mmol) and sodium hydrosulfite (7.0 g, 85% pure from Aldrich,40 mmol) in acetone and water (1:1, 20 mL) was refluxed for 3 h. Uponcooling, the volatile components (mainly acetone) were removed onrotavap, and the resulting mixture was subjected to filtration. Thesolid was thoroughly washed with EtOAc. The combined filtrate wasextracted with n-BuOH (4×), and the combined organic layer was washedwith saturated aq. NaHCO₃, dried (Na₂SO₄), filtered, concentrated, andthe resulting residue was purified by silica gel chromatography (elutedwith 5% MeOH/DCM, MeOH was pre-saturated with ammonia gas) to provide0.71 g of product as a pale yellow solid.

3-iodo-4-methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)Benzamide: 3-iodo-4-methylbenzoyl chloride (0.48 g, 1.7 mmol), preparedfrom the reaction of 3-iodo-4-methylbenzoic acid and SOCl₂ (aspreviously described), was added to a solution of4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)aniline (0.47 g,1.7 mmol), N,N-diisopropylethylamine (0.26 g, 2.0 mmol), and a catalyticamount of DMAP in THF (10 mL). After stirring at rt for 2 h, thereaction was quenched with water. EtOAc was added and the layersseparated. The combined organic layers were concentrated to dryness andpurified by silica gel chromatography (eluted with 5% MeOH/DCM, MeOH waspre-saturated with ammonia gas), to provide 0.51 g of product as anoff-white solid.

4-Methyl-N-[4-(4-methyl-piperazin-1-ylmethyl)-3-trifluoromethyl-phenyl]-3-trimethylsilanylethynyl-benzamide:3-iodo-4-methyl-N-[4-(4-methyl-piperazin-1-ylmethyl)-3-trifluoromethyl-phenyl]-benzamide(2.59 g, 5 mmol), Pd[(PPh₃)]₄ (289 mg, 0.25 mmol), Cul (71 mg, 0.375mmol) was placed in a schlenk flask. The flask was subjected to 3 cyclesof vacuum-refilling with N₂. To this mixture was added anhydrousN,N-diisopropylethylamine (1.1 mL, 6 mmol), DMF (5 mL), andtrimethylsilylacetylene (0.92 mL, 6.5 mmol). This solution was stirredat it for 24 h. Water and EtOAc were added to the reaction mixture tofacilitate the extraction. The combined organic layers were dried overNa₂SO₄, filtered, and then concentrated on a rotavap and the residue waspurified on a silica gel column (eluent: 5% MeOH in CH₂Cl₂, MeOH waspre-saturated with ammonia gas) to give the desired product as a lightyellow solid in 82% yield (2.0 g).

3-Ethynyl-4-methyl-N-[4-(4-methyl-piperazin-1-ylmethyl)-3-trifluoromethyl-phenyl]-benzamide:To a solution of4-methyl-N-[4-(4-methyl-piperazin-1-ylmethyl)-3-trifluoromethyl-phenyl]-3-trimethylsilanylethynyl-benzamide(2.0 g, 4.1 mmol) in THF (15 mL) was added 5 mL of TBAF in THF (1.0M).After stirring at it for 1 h, the mixture was partitioned between H₂Oand EtOAc. The combined organic layers were dried over Na₂SO₄, filtered,and then concentrated on a rotavap and the residue was purified on asilica gel column (eluent: 10% MeOH in CH₂Cl₂, MeOH was pre-saturatedwith ammonia gas) to give the desired product as a light yellow solid in78% yield (1.33 g).

1H-Pyrazole-1-N-methyl carboxamide: To 4-iodopyrazole (3 g, 15.5 mmol)in dry CH₂Cl₂ (20 mL) was added p-nitrophenyl chloroformate (3.43 g, 17mmol) in CH₂Cl₂ (30 mL) followed by TEA (2.5 mL, 18.6 mmol). Thismixture was stirred at rt for 2 h. The mixture was then diluted withexcess CH₂Cl₂ (60 mL) and washed with 10% aq. NaHCO₃. The organic layerwas dried over Na₂SO₄ and the solvent was evaporated to yield a solidwhich was recrystallized from Et₂O to furnish a white solid (5 g). Theresulting material (1 g, 2.79 mmol) in THF (12 mL) was treated with asolution of MeNH₂ (2 mL, 2M in THF) for 10 minutes. The organic layerwas evaporated to a solid which was dissolved in DCM and washed with 5%NaOH. The organic layer was dried, filtered and triturated with ether toafford the product as colorless shiny flakes (0.55 g).

3-(1H-Pyrazole-1-carboxamide-N-methyl)-4-methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)benzamide:3-Ethynyl-4-methyl-N-[4-(4-methyl-piperazin-1-ylmethyl)-3-trifluoromethyl-phenyl]-benzamide(91 mg, 0.22 mmol), 1H-Pyrazole-1-N-methyl carboxamide (0.2 mmol),Pd[(PPh₃)]₄ (11.6 mg, 0.01 mmol), Cul (2.9 mg, 0.015 mmol) was placed ina vial capped with rubber septa. This vial was subjected to 3 cycles ofvacuum-refilling with N₂. To this mixture was added anhydrousN,N-diisopropylethylamine (0.1 mL, 0.6 mmol) and DMF (1.0 mL). Theresulting solution was stirred at 80° C. for 24 h. After the reactionmixture was cooled, water and EtOAc were added to facilitate theextraction. The combined organic layers were dried over Na₂SO₄,filtered, and then concentrated on a rotavap and the residue waspurified on a silica gel column (eluent: 10% MeOH in CH₂Cl₂, MeOH waspre-saturated with NH₃ gas) to give the desired product as a lightyellow solid in 56% yield (63.0 mg): MS (M+H)⁺ 538.

Example 21Methyl-5-(2-methyl-5-[4-(4-methyl-piperazin-1-ylmethyl)-3-trifluoromethyl-phenylcarbamoyl]-phenylethynyl)-1H-imidazole-2-carboxylicacid amide

5-Ethynyl-1-methyl-1H-imidazole-2-carboxylic acid amide: To a solutionof 1-methyl-5-trimethylsilanylethynyl-1H-imidazole (1.78 g, 10 mmol) inTHF (30 mL) was slowly added a solution of n-BuLi in hexanes (2.5M, 4.4mL) at −78° C. and the resulting suspension was stirred at the sametemperature for 1 h. Trimethylsilyl isocyanate (from Aldrich, 85% pure,11.76 mmol, 1.6 mL) was slowly added to this suspension at −78° C. Afterstirring at −78° C. for another 30 minutes the reaction mixture wasallowed to warm up slowly by removing the cooling bath. Water (2 mL) andMeOH (1 mL) was added to quench the reaction and the mixture was stirredovernight. More water and EtOAc was added to facilitate the extraction.The combined organic layers were dried over Na₂SO₄, filtered, and thenconcentrated on a rotavap and the residue was purified on a silica gelcolumn (eluent: 40-60% EtOAc in hexanes) to give the desired product asa white solid in 26% yield (387 mg).

1-Methyl-5-(2-methyl-5-[4-(4-methyl-piperazin-1-ylmethyl)-3-trifluoromethyl-phenylcarbamoyl]-phenylethynyl)-1H-imidazole-2-carboxylicacid amide: 5-Ethynyl-1-methyl-1H-imidazole-2-carboxylic acid amide (33mg, 0.22 mmol),3-iodo-4-methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)benzamideas prepared in example 1 (103.4 mg, 0.2 mmol), Pd[(PPh₃)₄] (11.6 mg, 5mol %), and Cul (2.9 mg, 7.5 mmol %) were placed in a vial with rubberseptum. The mixture underwent 3 cycles of vacuum/filling with N₂, andDMF (1.5 ml) and N,N-diisopropylethylamine (53 μL, 0.3 mmol) were added.The mixture was stirred at rt for 16 h, and the reaction was quenchedwith H₂O. EtOAc and more water were added for extraction. The combinedorganic layer was dried (Na₂SO₄), filtered, concentrated, and theresulting residue was purified by silica gel chromatography (eluent: 5%MeOH in methylene chloride, MeOH was pre-saturated with ammonia gas),giving the titled compound as an off-white solid (65%, 70 mg): MS (M+H)⁺539.

Example 31Methyl-5-(2-methyl-5-[3-(4-methyl-imidazol-1-yl)-5-trifluoromethyl-phenylcarbamoyl]-phenylethynyl)-1H-imidazole-2-carboxylicacid amide

The title compound was made as for example 2 using5-ethynyl-1-methyl-1H-imidazole-2-carboxylic acid amide and3-iodo-4-methyl-N-(3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)benzamide:MS (M+H)⁺ 507.

3-(4-Methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)benzenamine: Asuspension of 3-bromo-5-(trifluoromethyl)aniline (4.8 g, 20 mmol),4-methylimidazole (1.97 g, 24 mmol), potassium carbonate (3.04 g, 22mmol), Cul (0.57 g, 3 mmol), and 8-hydroxyquinoline (0.44 g, 3 mmol,) indry DMSO (20 mL) in a pressure tube was degassed by bubbling N₂ into thesuspension for 10 minutes while stirring. The tube was sealed tightly.The mixture was heated at 120° C. (oil bath temperature) for 15 h. Themixture was cooled down to 45-50° C. and 14% aq. NH₄OH (20 mL) wasadded. The mixture was maintained at this temperature for 1 h. Aftercooling to rt, water and ethyl acetate were added. The aqueous layer wasextracted with ethyl acetate and the combined organic layers were passedthrough a short silica gel column to remove most of green/blue Cu salts.The filtrate was dried over sodium sulfate and concentrated on arotavap. The crude product was recrystallized from EtOAc/hexanes, givingpure pale yellow needles. The mother liquor was concentrated and theresidue was purified on silica gel column (5% methanol/methylenechloride), yielding a second crop as pale yellow needles.

3-iodo-4-methyl-N-(3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)Benzamide: 3-iodo-4-methylbenzoic acid (2.62 g, 10 mmol) was refluxed inSOCl₂ (10 mL) for 1 h. The volatile components were removed on a rotavapand the residue was dissolved in benzene (10 mL), concentrated todryness on a rotavap and further dried under vacuum. The resulting acylchloride was added to a solution3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)benzenamine (2.46 g,10.2 mmol), N,N-diisopropylethylamine (1.56 g, 12 mmol), and a catalyticamount of DMAP in THF (20 mL). After stirring at rt for 2 h, thereaction was quenched with water. EtOAc was added and the layersseparated. The combined organic layers were concentrated to dryness andused without purification in the coupling step.

Example 45-[5-(3-imidazol-1-yl-5-trifluoromethylphenylcarbamoyl)-2-methyl-phenylethynyl]-1-methyl-1H-imidazole-2-carboxylicacid amide

This was made as for example 2 using5-ethynyl-1-methyl-1H-imidazole-2-carboxylic acid amide andN-(3-(1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)-3-iodo-4-methylbenzamide:MS (M+H)⁺ 493.

3-(1H-imidazol-1-yl)-5-(trifluoromethyl)aniline: A mixture of3-Amino-5-bromobenzotrifluoride (4.0 g, 0.0167 mol), 8-hydroxy quinoline(0.362 g, 0.0025 mol), Cul (0.476 g. 0.025 mol), imidazole (1.36 g,0.0199 mol), and potassium carbonate (2.52 g, 0.0183 mol) in 17 mL ofDMSO (degassed with argon for −10 min) was heated at 120° C. under anatmosphere of argon for 15 h; the HPLC indicated no starting material. A14% aqueous solution of ammonium hydroxide was added to the cooledmixture and this was stirred for 1 h at ambient temperature. Water (50mL) and EtOAc (200 mL) were added and the aqueous layer was extractedwith EtOAc (3×30 mL). The combined organic layers were dried over Na₂SO₄and concentrated. The crude product was purified by silica gel flashchromatography (eluted with EtOAc/hexanes) to provide 2.51 g of product.

N-(3-(1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)-3-iodo-4-methylbenzamide:To 3-iodo-4-methylbenzoic acid (3.07 g, 0.0117 mol) was added thionylchloride (10 mL) and refluxed for 2 h. The excess thionyl chloride wascarefully removed and the resulting acid chloride was dried in vacuo for2 h. The residue was then dissolved in DCM (anhydrous, 25 mL) and cooledon ice. To the cooled solution was added3-(1H-imidazol-1-yl)-5-(trifluoromethyl)aniline (3.46 g, 0.0152 mol) inDCM followed by the dropwise addition of diisopropylethylamine (8.2 mL,0.047 mol). This was stirred at ambient temperature for 21 h. The whitesolid that separated was filtered and washed with water and dried toprovide 4.65 g of product. Additional product could be obtained from thefiltrate following concentration and purification by silica gel flashchromatography in EtOAc/hexanes.

Example 53-[(2-amino-1,3-thiazol-5yl)ethynyl]-4-methyl-N-[3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl]benzamide

tert-butyl {5-[(trimethylsilyl)ethynyl]-1,3-thiazol-2-yl}carbamate: Amixture of tert-butyl (5-bromo-1,3-thiazol-2-yl)carbamate (2.79 g, 10mmol), ethynyltrimethylsilane (1.27 g, 13 mmol), Pd(PPh₃)₄ (578 mg, 0.5mmol), Cul (143 mg, 0.75 mmol), and diisopropylethylamine (1.94 g, 15mmol) in DMF (10 mL) was heated at 50° C. overnight under an atmosphereof N₂. Upon cooling to ambient temperature, the reaction mixture wasconcentrated and the crude product was purified by silica gel flashchromatography (eluted with 20% EtOAc/hexanes) to give a yellow solid(2.55 g, 86%).

tert-butyl{5-[(2-methyl-5-{[3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl]carbamoyl}phenyl)ethynyl]-1,3-thiazol-2-yl}-carbamate:A mixture of tert-butyl{5-[(trimethylsilyl)ethynyl]-1,3-thiazol-2-yl}carbamate (166 mg, 0.56mmol), 3-iodo-4-methyl-N-(3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)benzamide (247 mg. 0.51 mmol), Pd(PPh₃)₄ (29 mg,0.025 mmol), Cul (7.1 mg, 0.0375 mmol), TBAF (1.0 M in THF, 0.62 mL,0.62 mmol), and diisopropylethylamine (0.14 mL, 0.78 mmol) in 3.0 mL ofDMF was stirred at ambient temperature overnight under an atmosphere ofN₂. The reaction was quenched with H₂O. EtOAc and more water were addedfor extraction. The combined organic layers were dried (Na₂SO₄),filtered, concentrated, and the resulting residue was purified by silicagel flash chromatography (eluted with 10% MeOH in CH₂Cl₂) to give thedesired product as a brownish solid (216 mg, 73%).

3-[(2-amino-1,3-thiazol-5-yl)ethynyl]-4-methyl-N-[3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl]benzamide:The solution of tert-butyl{5-[(2-methyl-5-{[3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl]carbamoyl}phenyl)ethynyl]-1,3-thiazol-2-yl}carbamate(216 mg) in TFA/CH₂Cl₂ (10 mL, 1:1) was stirred at room temperature for1 hr. The volatile components were removed on rotavap and the residuewas partitioned between EtOAc and aq. NaHCO₃. The combined organic layerwas dried (Na₂SO₄), filtered, concentrated, and the resulting residuewas purified by silica gel flash chromatography (eluted with 10% MeOH inCH₂Cl₂) to give the desired product as a brownish solid (170 mg, 95%):482 m/z (M+H).

Example 63-[(2-(acetylamino)-1,3-thiazol-5-yl]ethynyl)-4-methyl-N-[3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl]benzamide

A solution of3-[(2-amino-1,3-thiazol-5-yl)ethynyl]-4-methyl-N-[3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl]benzamide(100 mg) in Ac₂O (10 mL) was heated at 130° C. for 2 hr. Upon cooling toroom temperature the volatile components were removed under vacuum andthe residue was partitioned between EtOAc and aq. NaHCO₃. The combinedorganic layers were dried (Na₂SO₄), filtered, concentrated, and theresulting residue was purified by silica gel flash chromatography(eluted with 10% MeOH in CH₂Cl₂) to give the desired product as abrownish solid (105 mg, 96%): 524 m/z (M+H).

Example 7 Potential Synthesis of4-[(5-([3-(2-[(dimethylamino)methyl]-1H-imidazol-1yl)-5-trifluoromethyl)phenyl]carbamoyl)-2-methylphenyl)ethynyl]-N-methyl-1H-pyrazole-1-carboxamide

The title compound can be synthesized from1H-pyrazole-1-N-methylcaboxamide andN-[3-{2-[(dimethylamino)methyl]-1H-imidazol-1-yl}-5-(trifluoromethyl)phenyl]-3-ethynyl-4-methylbenzamide in a manner similar to that described forExample 1.

1-(1H-imidazol-2-yl)-N,N-dimethylmethanathine: To a two-neckedround-bottomed flask equipped with a reflux condenser and apressure-equalizing addition funnel, was added 2-imidazolecarboxaldehyde(6 g, 62.5 mmol) in MeOH (60 mL). To this suspension (ambienttemperature) was added a solution of dimethylamine (40% aqueous, 60 mL)at a fast dropping rate (20 min). After the addition was complete, solidsodium borohydride (7 g, 186.8 mmol,) was CAUTIOUSLY added portionwiseover 45 min. Foaming occurred after each portion, and the internaltemperature was allowed to maintain −50° C. without external cooling.The reaction mixture was then heated to 65° C. for 3 h and allowed tocool to ambient temperature for overnight. The reaction contents wereconcentrated in vacuo and the resultant residue was taken up in EtOAc(2×30 mL) washed with brine and with CHCl₃ (4×100 mL). The EtOAc extractwas discarded. The CHCl₃ extract was dried over (NaSO₄), filtered, andconcentrated in vacuo to give 3.7 g of the desired product as a waxysolid.

3-(2-((Dimethylamino)methyl)-1H-imidazol-1-yl)-5-(trifluoromethyl)aniline:3-Amino-5-bromobenzotrifluoride (6 g, 25 mmol) and1-(1H-imidazol-2-yl)-N,N-dimethylmethanamine (3.7 g, 29.8 mmol) weredissolved in anhydrous DMSO (25 mL). To this was added Cul (0.95 g, 7.5mmol), 8-hydroxy quinoline (0.72 g, 7.5 mmol) and K₂CO₃ (6.9 g, 50mmol). The mixture was stirred vigorously and degassed with N₂ for 15minutes. The flask was then equipped with a condenser and heated at 120°C. for 18 h. The resultant heterogeneous mixture was cooled to rt,poured into 14% aq. NH₄OH (100 mL) and extracted with EtOAc (3×300 ml).The combined extracts were dried over NaSO₄ and concentrated in vacuo.The residue was chromatograhed over silica gel eluting with MeOH/DCM(5:95) to furnish 3.5 g of the desired product as a tan coloredmaterial: 285 m/z (M+H).

N-(3-(2((dimethylamino)methyl)-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)-3-iodo-4-methylbenzamide:3-iodo-4-methylbenzoyl chloride (2.2 g, 7.88 mmol), dissolved inanhydrous THF (13 mL), was added dropwise to a solution of3-(2-((dimethylamino)methyl)-1H-imidazol-1-yl)-5-(trifluoromethyl)aniline(1.5 g, 5.5 mmol), DIPEA (2.1 mL, 11.8 mmol) in THF (30 mL) at ˜5° C.The resultant solution was stirred at ambient temperature overnight. Thesolvent was removed in vacuo and the crude residue was redissolved inCH₂Cl₂ and washed with 1N NaOH. The organic layer was then washed withwater, and brine then dried over NaSO₄ before being concentrated invacuo. The brown colored residue was then triturated in a mixture ofhexanes/DCM to precipitate 1.4 g of the desired product as an off-whitepowder: 529 m/z (M+H).

Potential synthesis ofN-[3-(2-[(dimethylamino)methyl]-1H-imidazol-1yl)-5-(trifluoromethyl)phenyl]-4-methyl-3((trimethylsilyl)ethynyl)benzamide:N-(3-(2-((dimethylamino)methyl)-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)-3-iodo-4-methylbenzamide(5 mmol), Pd[(PPh₃)]₄ (289 mg, 0.25 mmol), Cul (71 mg, 0.375 mmol) areplaced in a schlenk flask. The flask is subjected to 3 cycles of vacuum-refilling with N₂. To this mixture is added anhydrousN,N-diisopropylethylamine (1.1 mL, 6 mmol), DMF (5 mL), andtrimethylsilylacetylene (0.92 mL, 6.5 mmol). This solution is stirred atrt for 24 h. Water and EtOAc are added to the reaction mixture tofacilitate the extraction. The combined organic layers are dried overNa₂SO₄, filtered, and then concentrated on a rotavap and the residue ispurified on a silica gel column (eluent: 5% MeOH in CH₂Cl₂, MeOH waspre-saturated with ammonia gas) to give the desired product.

Potential Synthesis ofN-[3-(2-[(dimethylamino)methyl]-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl]-3-ethynyl-4-methylbenzamide:To a solution ofN-[3-(2-[(dimethylamino)methyl]-1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl]-4-methyl-3-[(trimethylsilyl)ethynyl]benzamide(4.1 mmol) in THF (15 mL) is added 5 mL of TBAF in THF (1.0M). Afterstirring at rt for 1 h, the mixture is partitioned between H₂O andEtOAc. The combined organic layers are dried over Na₂SO₄, filtered, andthen concentrated on a rotavap and the residue is purified on a silicagel column (eluent: 10% MeOH in CH₂Cl₂, MeOH was pre-saturated withammonia gas) to give the desired product.

Example 8 Potential Synthesis ofN-(3-chloro-4-[(4-methylpiperazin-1-yl)methyl]phenyl)-4-methyl-3-[(1-methyl-1H-imidazol-5-yl)ethynyl]benzamide:

The title compound can be synthesized according to Example 2, from5-ethynyl-1-methyl-1H-imidazole (prepared from deprotection of1-methyl-5-trimethylsilanylethynyl-1H-imidazole) andN-(3-chloro-4-((4-methylpiperazin-1-yl)methyl)phenyl)-3-iodo-4-methylbenzamide.

1-(Bromomethyl)-2-chloro-4-nitro-benzene: A suspension of2-chloro-4-nitrotoluene (10.0 g, 58.3 mmol), N-bromosuccinimide (NBS,10.9 g, 61.2 mmol), and 2,2′-azobis(2-methylpropionitrile) (AIBN, 0.29g, 1.75 mmol) in 120 mL of CCl₄ was heated at reflux under an atmosphereof N₂ for 12 h. The reaction mixture was cooled to ambient temperature,and the solid was filtered and washed with EtOAc. The combined filtratewas washed with aq. NaHCO₃, dried over Na₂SO₄, filtered, concentrated onrotovap, and further dried under vacuum. ¹H NMR indicated the ratio ofdesired product to unreacted 2-chloro-4-nitrotoluene to be 50:50. Thismaterial was used directly in the next step.

1-(2-Chloro-4-nitrobenzyl)-4-methylpiperazine: To a solution of crude1-(bromomethyl)-2-chloro-4-nitro-benzene (29.1 mmol; 50% pure) in 30 mLof DCM was added Et₃N (4.2 mL, 30 mmol) and 1-methylpiperazine (3.4 mL,30 mmol). After stirring for 3 h at ambient temperature, aq. NaHCO₃ wasadded and the mixture was extracted with DCM. The combined organic layerwas dried over Na₂SO₄, filtered, concentrated, and the resulting residuewas purified by silica gel chromatography (eluted with 5% MeOH/DCM) toprovide 6.80 g of product as a dark yellow oil.

3-Chloro-4-((4-methylpiperazin-1-yl)methyl)aniline: To a solution of1-(2-chloro-4-nitrobenzyl)-4-methylpiperazine (0.96 g, 3.6 mmol) inMeOH/water (4:1, 50 mL) was added 1.80 g (33.7 mmol) of NH₄Cl and 1.47 g(26.3 mmol) of Fe dust and the mixture heated at reflux under anatmosphere of N₂ for 2 h (HPLC indicated no progress). To this was added4 mL of glacial acetic acid and the mixture heated at reflux for anadditional 2 h. The reaction mixture was cooled to ambient temperature,filtered, and the filtrate concentrated. The residue was partitionedbetween EtOAc and saturated aq. NaHCO₃, the separated aqueous layer wasextracted with EtOAc, and the combined organics washed with brine anddried over Na₂SO₄. Upon concentration, the crude product was purified bysilica gel chromatography (eluted with 5-7% MeOH/DCM; silica geldeactivated with 1% triethylamine/DCM) to provide 0.53 g of product.

Alternative Potential Synthesis ofN-(3-chloro-4-[(4-methylpiperazin-1-yl)methyl]phenyl)-4-methyl-3-[(1-methyl-1H-imidazol-5-yl)ethynyl]benzamide

5-ethynyl-1-methyl-1H-imidazole: To a solution of1-methyl-5-trimethylsilanylethynyl-1H-imidazole (1.39 mol) in 10× volumeof Ethyl acetate and 1.5× volume of Methanol is added two and a halfequivalents of potassium carbonate at ambient temperature and thesolution stirred for 1 hour. Potassium carbonate is filtered off and theorganic stream is washed with water and with saturated sodium chloridesolution (two or more times). Aqueous phases can be combined andre-extracted with ethyl acetate. Organic streams can than be combinedand concentrated under vacuum to about 0.5 L. Solids can be allowed toprecipitate out upon concentration. Slurry is cooled, e.g. to about −5°C., stored overnight, filtered, and washed with about 0.3 L of coldethyl acetate. The solids can then be dried under vacuum.

4-methyl-3-[(1-methyl-1H-imidazol-5-yl)ethynyl]benzoic acid can beprepared in a manner similar to that described above for the Sonogashirareaction. 5-ethynyl-1-methyl-1H-imidazole and 3-iodo-4-methylbenzoicacid can be used as coupling partners. Alternatively, the solvent (DMF)can be replaced by ethyl acetate and the base (Hunig base) can bereplaced by triethylamine. The product can be isolated by filtration ofthe crude reaction mixture. The filter cake is washed sequentially witha solvent such as ethyl acetate and then water, then dried in a vacuumoven. Further purification can be achieved by slurrying the solids inwater adjusted to pH 3 with the addition of concentrated HCl. Afterfiltration and water wash, the product can be dried in a vacuum oven.

N-(3-chloro-4-[(4-methylpiperazin-1-yl)methyl]phenyl)-4-methyl-3-[(1-methyl-1H-imidazol-5-yl)ethynyl]benzamide:4-methyl-3-[(1-methyl-1H-imidazol-5-yl)ethynyl]benzoic acid (18 mmol) isdissolved in methylene chloride (100 mL). To this solution is added 3equivalents of 4-methylmorpholine (NMM) followed by 1.05 equivalents ofoxalyl chloride. After stirring at ambient temperature for 30 minutes,0.8 equivalents of 3-Chloro-4-((4-methylpiperazin-1-yl)methyl)aniline(prepared as above) is added along with 5 mole % of DMAP. Afterinitially stirring at ambient temperature, the mixture is brought toreflux and stirred overnight. After 16 h an additional 0.2 equivalentsof the aniline is added, bringing the total charge to 1 equivalent. Themixture can then be stirred for an additional 2 h, quenched with water,and the layers separated. The aqueous layer can be extracted withmethylene chloride (2×50 mL) and the combined extracts can be washedwith water. The combined methylene chloride layers can then beevaporated and the residue dissolved in 100 mL of ethyl acetate (20 mL).After standing for 1 h, the product is allowed to crystallize. Themixture is cooled, e.g. to 0° C., filtered, and the solid product iswashed with cold ethyl acetate.

Example 9 Potential Synthesis of3-[(2-amino-1,3-thiazol-5-yl)ethynyl]-N-(3-cyclopropyl-4-[(4-methylpiperazin-1-yl)methyl]phenyl)-4-methylbenzamide

The title compound can be synthesized from tert-butyl(5-ethynyl-1,3-thiazol-2-yl)carbamate andN-(3-cyclopropyl-4-((4-methylpiperazin-1-yl)methyl)phenyl)-3-iodo-4-methylbenzamidein a manner similar to that described for Example 2 (nitro reductionperformed in a manner similar to that described for Example 8; 0.25M inMeOH/10% AcOH). t-BOC deprotection can be performed after coupling asdescribed in Example 5.

Potential Synthesis of tert-butyl (5-ethynyl-1,3-thiazol-2-yl)carbamate:To a solution of tert-butyl(5-[(trimethylsilyl)ethynyl]-1,3-thiazol-2-yl)carbamate (prepared as inExample 5, 1.39 moil) in 10× volume of Ethyl acetate and 1.5× volume ofMethanol is added two and a half equivalents of potassium carbonate atambient temperature and the solution stirred for 1 hour. Potassiumcarbonate is filtered off and the organic stream is washed with waterand with saturated sodium chloride solution (two or more times). Aqueousphases can be combined and re-extracted with ethyl acetate. Organicstreams can then be combined and concentrated under vacuum to about 0.5L. Solids can be allowed to precipitate out upon concentration. Slurryis cooled, e.g. to about −5° C., stored overnight, filtered, and washedwith about 0.3 L of cold ethyl acetate. The solids can then be driedunder vacuum.

1-(2-Cyclopropyl-4-nitrobenzyl)-4-methylpiperazine: A mixture of1-(2-bromo-4-nitrobenzyl)-4-methylpiperazine (0.94 g, 3.0 mmol), 0.77 g(9.0 mmol) of cyclopropylboronic acid, 0.057 g (0.30 mmol) of Pd(OAc)₂,2.87 g (13.5 mmol) of K₃PO₄, and 0.168 g (0.60 mmol) oftricyclohexylphosphine in 18 mL of toluene/water (5:1) was heated atreflux under an atmosphere of N₂ for 19 h. The reaction mixture wasconcentrated and the crude product was purified by silica gelchromatography (eluted with 5% MeOH/DCM; MeOH was pre-saturated withammonia gas) to provide 0.80 g of product.

Example 10 Potential Synthesis of1-methyl-5-((2-methyl-5[((4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl)carbonyl)amino]phenyl)ethynyl)-1H-imidazole-2-carboxamide

The title compound can be synthesized from5-ethynyl-1-methyl-1H-imidazole-2-carboxylic acid amide andN-(3-iodo-4-methylphenyl)-4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)benzamidein a manner similar to that described for Example 2.5-ethynyl-1-methyl-1H-imidazole-2-carboxylic acid amide is prepared asin Example 2.

N-(3-iodo-4-methylphenyl)-4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)benzamide:To a flask containing 1.0 g (2.67 mmol) of4-[(4-methyl-1-piperazinyl)methyl]-3-(trifluoromethyl)-benzoic acid (CAS#859027-02-4; prepared according to Asaki, T. et al. Bioorg. Med. Chem.Lett. (2006), 16, 1421-1425), 0.62 _(g) (2.67 mmol) of3-iodo-4-methylaniline, 0.77 g (4.0 mmol) ofN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDAC), and0.43 g (3.2 mmol) of N-hydroxybenzotriazole monohydrate (HOBt.H₂O) wasadded 5 mL of DCM and 5 mL of triethylamine. The solution was stirred atambient temperature under an atmosphere of N₂ for 3 days, concentrated,and the crude product purified by silica gel chromatography (eluted with100% EtOAc then 10% MeOH/EtOAc), to provide 0.69 g of product as a whitesolid.

Example 11 Potential Synthesis of5-[(5-[(4-[((3R)-3(dimethylamino)pyrrolidin-1-yl]methyl)-3-(trifluoromethyl)phenyl]carbamoyl)-2-methylphenyl)ethynyl]-1-methyl-1H-imidazole-2-carboxamide

The title compound can be synthesized from5-ethynyl-1-methyl-1H-imidazole-2-carboxylic acid amide and(R)-N-(4-((3-(Dimethylamino)pyrrolidin-1-yl)methyl)-3-(trifluoromethyl)phenyl)-3-iodo-4-methylbenzamidein a manner similar to that described for Example 2.5-ethynyl-1-methyl-1H-imidazole-2-carboxylic acid amide is prepared asin Example 2.

1-(Bromomethyl)-4-nitro-2-(trifluoromethyl)benzene: A suspension of2-methyl-5-nitrobenzotrifluoride (3.90 g, 19 mmol), N-bromosuccinimide(NBS, 3.56 g, 20 mmol), and 2,2′-azobis(2-methylpropionitrile) (AIBN,0.094 g, 0.6 mmol) in 40 mL of CCl₄ was heated at reflux under N₂ for 16h. HPLC indicated ca. 50% conversion. Additional NBS (10 mmol) and AIBN(0.6 mmol) were added and the mixture was heated at reflux for another14 h. HPLC indicated ca. 80% conversion. The reaction mixture was cooledto ambient temperature, and the solid was filtered and washed withEtOAc. The combined filtrate was washed with aq. NaHCO₃, dried overNa₂SO₄, filtered, concentrated on rotovap, and further dried undervacuum. ¹H NMR indicated the ratio of desired product to unreacted2-methyl-5-nitrobenzotrifluoride to be 75:25. This material was useddirectly in the next step.

(R)-N,N-Dimethyl-1-(4-nitro-2-(trifluoromethyl)benzyl)pyrrolidin-3-amine:To a solution of crude1-(bromomethyl)-4-nitro-2-(trifluoromethyl)benzene (17.5 mmol, 75% pure)in 40 mL of DCM was added Et₃N (2.69 mL, 19.3 mmol) and(R)-(+)-3-(dimethylamino)pyrrolidine (2.0 g, 17.5 mmol). After stirringovernight at ambient temperature under an atmosphere of N₂, the reactionsolution was concentrated, added aq. NaHCO₃ (100 mL), and the resultingmixture extracted with DCM (4×50 mL). The combined organic layer wasdried over Na₂SO₄, filtered, concentrated, and the resulting residue waspurified by silica gel chromatography (eluted with 0-10% MeOH/DCM) toprovide 3.35 g of product as a yellow oil.

(R)-1-(4-Amino-2-(trifluoromethyl)benzyl)-N,N-dimethylpyrrolidin-3-amine:To a solution of(R)-N,N-dimethyl-1-(4-nitro-2-(trifluoromethyl)benzyl)pyrrolidin-3-amine(1.20 g, 3.79 mmol) in 20 mL of wet EtOH was added 0.26 g of Pd/C (10%Pd on C) and the mixture shaken in a Parr apparatus (pressure reactionvessel purged thoroughly with H₂ and pressure regulated at 45 psithroughout) for 2-3 h. The reaction mixture was filtered through a smallpad of celite, washed with EtOAc, and the combined organics concentratedto provide a quantitative yield of a light yellow oil. This material wasused directly in the next step.

(R)-N-(4-((3-(Dimethylamino)pyrrolidin-1-yl)methyl)-3-(trifluoromethyl)phenyl)-3-iodo-4-methylbenzamide:To a cooled (0° C.) solution of(R)-1-(4-amino-2-(trifluoromethyl)benzyl)-N,N-dimethylpyrrolidin-3-amine(3.79 mmol) in 14 mL DCM, under an atmosphere of N₂, was added3-iodo-4-methylbenzoyl chloride (1.17 g, 4.17 mmol; CAS #52107-98-9,prepared from the reaction of 3-iodo-4-methylbenzoic acid and SOCl₂)followed by dropwise addition of N,N-diisopropylethylamine (2.64 mL,15.2 mmol). After stirring to ambient temperature over 1.5 h, thereaction mixture was concentrated and the crude product was purified bysilica gel chromatography (eluted with 0-8% MeOH/DCM; MeOH waspre-saturated with ammonia gas), to provide 0.71 g of product as a thickyellow oil.

Potential Synthesis of5-[(5-((4-(((3R)-3-(dimethylamino)pyrrolidin-1-yl)methyl)-3-(trifluoromethyl)phenyl]carbamoyl)-2-methylphenyl)ethynyl)-1-methyl-1H-imidazole-2-carboxamide: A mixture of 5-ethynylpyrimidne (0.34 mmol),0.150 g (0.28 mmol) of(R)-N-(4-((3-(dimethylamino)pyrrolidin-1-yl)methyl)-3-(trifluoromethyl)phenyl)-3-iodo-4-methylbenzamide, 0.016 g (0.014 mmol) of Pd(PPh₃)₄, 0.004 g (0.021 mmol)of Cul, and 0.09 mL (0.51 mmol) of N,N-diisopropylethylamine in 3.5 mLof DMF is stirred at ambient temperature, under an atmosphere of N₂, for3 days (reaction pushed to completion with additional equivalents ofreagents and heating to 80° C.). The reaction mixture is concentratedand the crude product is purified by silica gel chromatography (elutedwith 0-10% MeOH/DCM; MeOH was pre-saturated with ammonia gas) to providethe title compound.

Example 12: Biological Evaluation of Compounds

Compounds of this invention are evaluated in a variety of assays todetermine their biological activities. For example, the compounds of theinvention can be tested for their ability to inhibit various proteinkinases of interest. Some of the compounds tested displayed potentnanomolar activity against the following kinases: Abl, Abl T315I, Srcand FGFR. Furthermore, several of these compounds were screened forantiproliferative activity in BaF3 cells transfected with eitherwild-type Bcr-Abl or the Bcr-Abl T315I mutant and demonstrated activityin the range of 1-100 nM.

The compounds can also be evaluated for their cytotoxic or growthinhibitory effects on tumor cells of interest, e.g., as described inmore detail below and as shown above for some representative compounds.See e.g., WO 03/000188, pages 115-136, the full contents of which areincorporated herein by reference.

Some representative compounds are depicted below:

Kinase Inhibition

More specifically, the compounds described herein are screened forkinase inhibition activity as follows. Kinases suitable for use in thefollowing protocol include, but are not limited to: Abl, Lck, Lyn, Src,Fyn, Syk, Zap-70, Itk, Tec, Btk, EGFR, ErbB2, Kdr, Flt1, Flt-3, Tek,c-Met, InsR, and AKT.

Kinases are expressed as either kinase domains or full length constructsfused to glutathione S-transferase (GST) or polyHistidine tagged fusionproteins in either E. coli or Baculovirus-High Five expression systems.They are purified to near homogeneity by affinity chromatography aspreviously described (Lehr et al., 1996; Gish et al., 1995). In someinstances, kinases are co-expressed or mixed with purified or partiallypurified regulatory polypeptides prior to measurement of activity.

Kinase activity and inhibition can be measured by established protocols(see e.g., Braunwalder et al., 1996). In such cases, the transfer of³³PO₄ from ATP to the synthetic substrates poly(Glu, Tyr) 4:1 orpoly(Arg, Ser) 3:1 attached to the bioactive surface of microtiterplates is taken as a measure of enzyme activity. After an incubationperiod, the amount of phosphate transferred is measured by first washingthe plate with 0.5% phosphoric acid, adding liquid scintillant, and thencounting in a liquid scintillation detector. The IC50 is determined bythe concentration of compound that causes a 50% reduction in the amountof ³³P incorporated onto the substrate bound to the plate.

In one method, the activated kinase is incubated with a biotinylatedsubstrate peptide (containing tyrosine) with or without the presence ofa compound of the invention. After the kinase assay incubation period,excess kinase inhibitor is added to kill the kinase reaction along withEuropium-labeled anti-phosphotyrosine antibody (Eu-Ab) andAliophycocyanin-Streptavidin (SA-APC). The biotinylated substratepeptide (with or without phosphorylated Tyrosine) in solution binds tothe SA-APC via Biotin-Avidin binding. The Eu-Ab binds only to substratewith phosphorylated tyrosine. When the solution is excited at 615 nm,there is an energy transfer from the Europium to the APC when they arein close proximity (i.e. attached to the same molecule of biotinylatedand phosphorylated substrate peptide). The APC then fluoresces at awavelength of 665 nm. Excitation and emission take place in a WallacVictor² V plate reader where the plate is read fluorometrically andabsorbances at 615 and 665 nm are recorded. These data are thenprocessed by an Excel plate processor which calculates IC50s of testcompounds by converting the fluorescence into amounts of phosphorylatedsubstrate made and determining the concentration of test compound thatwould be required to inhibit the development of phosphorylated substrateby 50% (IC50).

Other methods relying upon the transfer of phosphate to peptide orpolypeptide substrate containing tyrosine, serine, threonine orhistidine, alone, in combination with each other, or in combination withother amino acids, in solution or immobilized (i.e., solid phase) arealso useful.

For example, transfer of phosphate to a peptide or polypeptide can alsobe detected using scintillation proximity, Fluorescence Polarization orhomogeneous time-resolved fluorescence. Alternatively, kinase activitycan be measured using antibody-based methods in which an antibody orpolypeptide is used as a reagent to detect phosphorylated targetpolypeptide.

For additional background information on such assay methodologies, seee.g., Braunwalder et al., 1996, Anal. Biochem. 234(I):23; Cleaveland etal., 1990, Anal Biochem. 190(2):249 Gish et al. (1995). Protein Eng.8(6):609 Kolb et al. (1998). Drug Discov. Toda V. 3:333 Lehr et al.(1996). Gene 169(2):27527-87 Seethaia et al. (1998). Anal Biochem.255(2):257 Wu et al. (2000).

IC50 values in the low nanomolar range have been observed for compoundsof this invention against various kinases, including Src, Abl and kdr.

Cell-Based Assays

Certain compounds of this invention have also been demonstratedcytotoxic or growth inhibitory effects on tumor and other cancer celllines and thus may be useful in the treatment of cancer and other cellproliferative diseases. Compounds are assayed for anti-tumor activityusing in vivo and in vitro assays which are well known to those skilledin the art. Generally, initial screens of compounds to identifycandidate anti-cancer drugs are performed in cellular assays. Compoundsidentified as having anti-proliferative activity in such cell-basedassays can then be subsequently assayed in whole organisms foranti-tumor activity and toxicity. Generally speaking, cell-based screenscan be performed more rapidly and cost-effectively relative to assaysthat use whole organisms. For purposes of this invention, the terms“anti-tumor” and “anti-cancer” activity are used interchangeably.

Cell-based methods for measuring antiproliferative activity are wellknown and can be used for comparative characterization of compounds ofthis invention. In general, cell proliferation and cell viability assaysare designed to provide a detectable signal when cells are metabolicallyactive. Compounds may be tested for antiproliferative activity bymeasuring any observed decrease in metabolic activity of the cells afterexposure of the cells to compound. Commonly used methods include, forexample, measurement of membrane integrity (as a measure of cellviability) (e.g. using trypan blue exclusion) or measurement of DNAsynthesis (e.g. by measuring incorporation of BrdU or 3H-thymidine).

Some methods for assaying cell proliferation use a reagent that isconverted into a detectable compound during cell proliferation.Particularly preferred compounds are tetrazolium salts and includewithout limitation MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;Sigma-Aldrich, St. Louis, Mo.), MTS(3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium),XTT(2,3-bis(2-Methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide),INT, NBT, and NTV (Bernas et al. Biochim Biophys Acta 1451(1):73-81,1999). Preferred assays utilizing tetrazolium salts detect cellproliferation by detecting the product of the enzymatic conversion ofthe tetrazolium salts into blue formazan derivatives, which are readilydetected by spectroscopic methods (Mosman. J. Immunol. Methods.65:55-63, 1983).

Generally, preferred methods for assaying cell proliferation involveincubating cells in a desired growth medium with and without thecompounds to be tested. Growth conditions for various prokaryotic andeukaryotic cells are well-known to those of ordinary skill in the art(Ausubel et al. Current Protocols in Molecular Biology. Wiley and Sons.1999; Bonifacino et al. Current Protocols in Cell Biology. Wiley andSons. 1999 both incorporated herein by reference). To detect cellproliferation, the tetrazolium salts are added to the incubated culturedcells to allow enzymatic conversion to the detectable product by activecells. Cells are processed, and the optical density of the cells isdetermined to measure the amount of formazan derivatives. Furthermore,commercially available kits, including reagents and protocols, areavailable for examples, from Promega Corporation (Madison, Wis.),Sigma-Aldrich (St. Louis, Mo.), and Trevigen (Gaithersburg, Md.).

More specifically, the cell proliferation assay we currently perform isusing CellTiter 96 AQueous One Solution Cell Proliferation assay kit(Promaga, Cat #G3581). This assay is a colorimetric method fordetermining the number of alive cells in proliferation or cytotoxicityassays. The assay utilizing terazolium salts detect cell proliferationby detecting the product of the enzymatic conversion of the tetrazoliumsalts into blue formazan derivatives, which can be measured by theabsorbance at 490 nm in a plate reader, Wallac Victor²V (PerkinElmer).

An example of cell-based assay is shown as below. The cell lines used inthe assay are Ba/F3, a murine pro-B cell line, which have been stablytransfected with full-length wild-type Bcr-Abl or Bcr-Abl with variouskinase domain point mutations (including T351I, Y253F, E255K, H396P,M351T etc) constructs. Parental Ba/F3 cell line is used as control.These cell lines were obtained from Brian J. Druker (Howard HughesMedical Institute, Oregon Health and Science University, Portland,Oreg., USA). Ba/F3 cell expressing Bcr-Abl or Bcr-Abl mutants weremaintained in PRMI 1640 growth medium with 200 μM L-gultamine, 10% FCS,penicillin (200 U/ml), and streptomycin (200 μg/ml). Parental Ba/F3cells were culture in the same medium supplemented with 10 ng/ml IL-3.

Parental Ba/F3 cells (supplemented with IL-3) or Ba/F3 cells expressingWT or mutant Bcr-Abl are plated in duplicate at 1×10⁴ cells/well in96-well plates with the compounds in different concentrations in themedia. The compounds are first dissolved and diluted in DMSO bypreparation of 4-fold dilution; next equal volumes of compounds withDMSO are transferred to medium and then transferred to cell plates. Thefinal compound concentrations start from 10 μM to 6 nM. DMSO at samepercentage is used as control. After compound was incubated with cellsfor 3 days, the numbers of active cells are measured using CellTiter 96AQueous One Solution Cell Proliferation assay kit following the kitinstruction. Basically, the tetrazolium salts are added to the incubatedcultured cells to allow enzymatic conversion to the detectable productby active cells. Cells are processed, and the optical density of thecells is determined to measure the amount of formazan derivatives.Mean+/−SD are generated from duplicated wells and reported as thepercentage absorbance of control. IC50s are calculated in best-fitcurves using Micorsoft Excel-fit software.

In addition, a wide variety of cell types may be used to screencompounds for antiproliferative activity, including the following celllines, among others: COLO 205 (colon cancer), DLD-1 (colon cancer),HCT-15 (colon cancer), HT29 (colon cancer), HEP G2 (Hepatoma), K-562(Leukemia), A549 (Lung), NCI-H249 (Lung), MCF7 (Mammary), MDA-MB-231(Mammary), SAOS-2 (Osteosarcoma), OVCAR-3 (Ovarian), PANC-1 (Pancreas),DU-145 (Prostate), PC-3 (Prostate), ACHN (Renal), CAKI-1 (Renal), MG-63(Sarcoma).

While the cell line is preferably mammalian, lower order eukaryoticcells such as yeast may also be used to screen compounds. Preferredmammalian cell lines are derived from humans, rats, mice, rabbits,monkeys, hamsters, and guinea pigs since cells lines from theseorganisms are well-studied and characterized. However, others may beused as well.

Suitable mammalian cell lines are often derived from tumors. Forexample, the following tumor cell-types may be sources of cells forculturing cells: melanoma, myeloid leukemia, carcinomas of the lung,breast, ovaries, colon, kidney, prostate, pancreas and testes),cardiomyocytes, endothelial cells, epithelial cells, lymphocytes (T-celland B cell), mast cells, eosinophils, vascular intimal cells,hepatocytes, leukocytes including mononuclear leukocytes, stem cellssuch as haemopoetic, neural, skin, lung, kidney, liver and myocyte stemcells (for use in screening for differentiation and de-differentiationfactors), osteoclasts, chondrocytes and other connective tissue cells,keratinocytes, melanocytes, liver cells, kidney cells, and adipocytes.Non-limiting examples of mammalian cells lines that have been widelyused by researchers include HeLa, NIH/3T3, HT1080, CHO, COS-1, 293T,WI-38 and CV1/EBNA-1.

Other cellular assays may be used which rely upon a reporter gene todetect metabolically active cells. Non-limiting examples of reportergene expression systems include green fluorescent protein (GFP), andluciferase. As an example of the use of GFP to screen for potentialantitumor drugs, Sandman et al. (Chem Biol. 6:541-51; incorporatedherein by reference) used HeLa cells containing an inducible variant ofGFP to detect compounds that inhibited expression of the GFP, and thusinhibited cell proliferation.

Compounds identified by such cellular assays as having anti-cellproliferation activity are then tested for anti-tumor activity in wholeorganisms. Preferably, the organisms are mammalian. Well-characterizedmammalians systems for studying cancer include rodents such as rats andmice. Typically, a tumor of interest is transplanted into a mouse havinga reduced ability to mount an immune response to the tumor to reduce thelikelihood of rejection. Such mice include for example, nude mice(athymic) and SCID (severe combined immunodeficiency) mice. Othertransgenic mice such as oncogene containing mice may be used in thepresent assays (see for example U.S. Pat. No. 4,736,866 and U.S. Pat.No. 5,175,383). For a review and discussion on the use of rodent modelsfor antitumor drug testing see Kerbel (Cancer Metastasis Rev.17:301-304, 1998-99).

In general, the tumors of interest are implanted in a test organismpreferably subcutaneously. The organism containing the tumor is treatedwith doses of candidate anti-tumor compounds. The size of the tumor isperiodically measured to determine the effects of the test compound onthe tumor. Some tumor types are implanted at sites other thansubcutaneous sites (e.g. intraperitoneal sites) and survival is measuredas the endpoint. Parameters to be assayed with routine screening includedifferent tumor models, various tumor and drug routes, and dose amountsand schedule. For a review of the use of mice in detecting antitumorcompounds see Corbett et al. (Invest New Drugs. 15:207-218, 1997;incorporated herein by reference).

Example 8: Pharmaceutical Compositions

Representative pharmaceutical dosage forms of the compounds of thisinvention (the active ingredient being referred to as “Compound”), areprovided for therapeutic or prophylactic use in humans:

(a) Tablet I mg/tablet Compound 100 Lactose Ph. Eur 182.75Croscarmellose sodium 12.0 Maize starch paste (5% w/v paste) 2.25Magnesium stearate 3.0

(b) Tablet II mg/tablet Compound 50 Lactose Ph. Eur 223.75Croscarmellose sodium 6.0 Maize starch 15.0 Polyvinylpyffolidone (5% w/vpaste) 2.25 Magnesium stearate 3.0

(c) Tablet III mg/tablet Compound 1.0 Lactose Ph. Eur 93.25Croscarmellose sodium 4.0 Maize starch paste (5% w/v paste) 0.75Magnesium stearate 1.0-76

(d) Capsule mg/capsule Compound 10 Lactose Ph. Eur 488.5 Magnesium 1.5

(e) Injection I (50 mg/ml) Compound 5.0% w/v 1M Sodium hydroxidesolution 15.0% w/v  0.1M Hydrochloric acid (to adjust pH to 7.6)Polyethylene glycol 400 4.5% w/v Water for injection to 100%

(f) Injection II (10 mg/ml) Compound 1.0% W/v Sodium phosphate BP 3.6%w/v O.1M Sodium hydroxide solution 15.0% v/v  Water for injection to100%

(g) Injection III (1 mg/ml, buffered to pH 6) Compound 0.1% w/v Sodiumphosphate BP 2.26% w/v  Citric acid 0.38% w/v  Polyethylene glycol 4003.5% w/v Water for injection to 100%

(h) Aerosol 1 mg/ml Compound 10.0 Sorbitan trioleate 13.5Trichlorofluoromethane 910.0 Dichlorodifluoromethane 490.0

(i) Aerosol II mg/ml Compound 0.2 Sorbitan trioleate 0.27Trichlorofluoromethane 70.0 Dichlorodifluoromethane 280.0Dichlorotetrafluoroethane 1094.0

(j) Aerosol III mg/ml Compound 2.5 Sorbitan trioleate 3.38Trichlorofluoromethane 67.5 Dichlorodifluoromethane 1086.0Dichlorotetrafluoroethane 191.6

(k) Aerosol IV mg/ml Compound 2.5 Soya lecithin 2.7Trichlorofluoromethane 67.5 Dichlorodifluoromethane 1086.0Dichlorotetrafluoroethane 191.6

(l) Ointment ml Compound 40 mg Ethanol 300 μl Water 300 μl1-Dodecylazacycloheptanone 50 μl Propylene glycol to 1 mlNote: These formulations may be prepared using conventional procedureswell known in the pharmaceutical art. The tablets (a)-(c) may be entericcoated by conventional means, if desired to provide a coating ofcellulose acetate phthalate, for example. The aerosol formulations(h)-(k) may be used in conjunction with standard, metered dose aerosoldispensers, and the suspending agents sorbitan trioleate and soyalecithin may be replaced by an alternative suspending agent such assorbitan monooleate, sorbitan sesquioleate, polysorbate 80, polyglycerololeate or oleic acid.

1. A compound of the Formula I, a tautomer, an individual isomer, amixture of isomers or a pharmaceutically acceptable salt, solvate orhydrate thereof:

wherein: Ring T represents a 5-membered monocyclic heteroaryl ring,comprising 1-3 heteroatoms selected from O, N and S and being optionallysubstituted with 1-3 R^(t) groups; Ring A represents a 5- or 6-memberedaryl or heteroaryl ring and is optionally substituted with 1-4 R^(a)groups; Ring B represents a 5- or 6-membered aryl or heteroaryl ring andis optionally substituted with 1-5 R^(b) groups; L¹ is selected fromNR¹C(O) and C(O)NR¹; At each occurrence, R^(a), R^(b) and R^(t), isindependently selected from the group consisting of halo, —CN, —NO₂,—R⁴, —OR², —NR²R³, —C(O)YR², —OC(O)YR², —NR²C(O)YR², —SC(O)YR²,—NR²C(═S)YR², —OC(═S)YR², —C(═S)YR², —YC(═NR³)YR², —YP(═O)(YR⁴)(YR⁴),—Si(R⁴)₃, —NR²SO₂R², —S(O)_(r)R², —SO₂NR²R³ and —NR²SO₂NR²R³, wherein Yis independently a bond, —O—, —S— or —NR³—; R¹, R² and R³ areindependently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocycle and heteroaryl;Alternatively, NR²R³ moiety may be 5- or 6-membered saturated, partiallysaturated or unsaturated ring, which can be optionally substituted andwhich contains 0-2 additional heteroatoms selected from N, O andS(O)_(r); each occurrence of R⁴ is independently selected from alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,heterocyclic and heteroaryl; each of the foregoing alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclic andheteroaryl moieties is optionally substituted; m is 0, 1, 2, 3 or 4; nis 0, 1, 2 or 3; p is 0, 1, 2, 3, 4 or 5; r is selected 0, 1 or
 2. 2. Acompound according to claim 1 wherein Ring T is a monocyclic 5-memberedring selected from:

and is optionally substituted on carbon or heteroatom with 1-3 R^(t)groups.
 3. A compound of claim 1 having the Formula:

wherein: Ring C represents a 5- or 6-membered heterocyclic or heteroarylring, comprising carbon atoms and 1-3 heteroatoms selected from O, N andS(O)_(r) and is optionally substituted on carbon or heteroatom(s) with1-5 R^(c) groups; R^(c), at each occurrence, is independently selectedfrom the group consisting of halo, ═O, ═S, —CN, —NO₂, —R⁴, —OR², —NR²R³,—C(O)YR², —OC(O)YR², —NR²C(O)YR², —SC(O)YR², —NR²C(═S)YR², —OC(═S)YR²,—C(═S)YR², —YC(═NR³)YR², —YP(═O)(YR⁴)(YR⁴), —Si(R⁴)₃, —NR²SO₂R²,—S(O)_(r)R², —SO₂NR²R³ and —NR²SO₂NR²R³, wherein Y is independently abond, —O—, —S— or —NR³—; R² and R³ are independently selected from H,alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,heterocyclic and heteroaryl; alternatively, NR²R³ moiety may be a 5- or6-membered saturated, partially saturated or unsaturated ring, which canbe optionally substituted and which contains 0-2 additional heteroatomsselected from N, O and S(O)_(r); each occurrence of R⁸ is independentlyselected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, aryl, heterocyclic and heteroaryl; each of the foregoingalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,heteroaryl and heterocycle moieties is optionally substituted; v is 0,1, 2, 3, 4 or 5 t is 0, 1, 2, 3, or
 4. 4. A compound according to claim3, wherein Rings A and B are aryl.
 5. A compound of claim 3 wherein RingC is a heteroaryl ring.
 6. A compound of claim 5 wherein Ring C is animidazole ring.
 7. A compound of claim 3 selected from the formulae IIa,IIb and IIc:


8. A compound of claim 7 wherein R^(t) is independently selected from—CH₃, or —C(O)NH₂, v is 1, n is 0 or 1, m is 1, t is 1, R^(a) is —CH₃,R^(b) is CF₃ and R^(c) is —CH₃.
 9. A compound of claim 1 having theFormula:

wherein: Ring D represents a 5- or 6-membered heterocyclic or heteroarylring, comprising carbon atoms and 1-3 heteroatoms independently selectedfrom N, O, S(O)_(r) and is optionally substituted with 1-5 R^(d) groups;L² is (CH₂)₂, O(CH₂)_(x), NR³(CH₂)_(x), S(CH₂)_(x) or(CH₂)_(x)NR³C(O)(CH₂)_(x) and the linkage unit can be used in eitherdirection; R^(d), at each occurrence, is selected from the groupconsisting of halo, ═O, ═S, —CN, —NO₂, —R⁴, —OR², —NR²R³, —C(O)YR²,—OC(O)YR², —NR²C(O)YR², —SC(O)YR², —NR²C(═S)YR², —OC(═S)YR², —C(═S)YR²,—YC(═NR³)YR², —YP(═O)(YR⁴)(YR⁴), —Si(R⁴)₃, —NR²SO₂R², —S(O)_(r)R²,—SO₂NR²R³ and —NR²SO₂NR²R³, wherein Y is independently a bond, —O—, —S—or —NR³—; R² _(and) R³ are independently selected from H, alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,heterocyclic and heteroaryl; alternatively, NR²R³ moiety may be a 5- or6-membered saturated, partially saturated or unsaturated ring, which canbe optionally substituted and which contains 0-2 additional heteroatomsselected from N, O and S(O)_(r); each occurrence of R4 is independentlyselected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, aryl, heterocyclic and heteroaryl; each of the foregoingalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,heteroaryl and heterocycle moieties is optionally substituted; w isselected from 0, 1, 2, 3, 4 or 5; x is 0, 1, 2 or 3; z is 1, 2, 3 or 4;and t is 0, 1, 2, 3, or
 4. 10. A compound according to claim 9 whereinRings A and B are aryl.
 11. A compound of claim 9 wherein Ring D is asubstituted or unsubstituted piperazine ring and L² is CH₂.
 12. Acompound of claim 9 wherein Ring D is a substituted or unsubstitutedheteroaryl.
 13. A compound of claim 11 selected from Formulae IIIa, IIIband IIIc:


14. A compound of claim 13 wherein R^(t) is independently selected from—CH₃ and —C(O)NH₂, n is 0 or 1, m is 1, t is 1, R^(a) is methyl, R^(b)is CF₃, one of R^(d) is selected from methyl and CH₂CH₂OH.
 15. A methodfor treating cancer in a mammal in need thereof, comprisingadministering to the mammal a therapeutically effective amount of acompound of any of claims 1-14 or a pharmaceutically acceptable salt,solvate or hydrate thereof.
 16. A composition comprising a compound ofany of claims 1-14 or a pharmaceutically acceptable salt, solvate orhydrate thereof and a pharmaceutically acceptable carrier, diluent orvehicle.